Composition and kit for detecting gonococcus, chlamydia trachomatis and ureaplasma urealyticum

Abstract

The invention discloses a composition and a kit for detecting gonococcus, chlamydia trachomatis and ureaplasma urealyticum. The composition comprises a primer pair and a probe corresponding to the primer pair, the primer pair comprises a primer pair 1 and a primer pair 2, wherein the primer pair 1 aims at gonococcus, and the nucleotide sequence of the primer pair 1 is shown as SEQ ID NO.01 and SEQ ID NO.02; the nucleotide sequences of the primer pair 2 aiming at the chlamydia trachomatis are as shown in SEQ ID NO.03 and SEQ ID NO.04; the nucleotide sequences of the primer pair 3 aiming at the ureaplasma urealyticum are as shown in SEQ ID NO.05 and SEQ ID NO.06; the probe comprises: a specific probe 1 aiming at gonococcus, the nucleotide sequence of which is as shown in SEQ ID NO.07; a specific probe 2 for the chlamydia trachomatis, wherein the nucleotide sequence of the specific probe 2 is as shown in SEQ ID NO.08; and the nucleotide sequence of the specific probe 3 aiming at the ureaplasma urealyticum is as shown in SEQ ID NO.09. The requirement of detection sensitivity can be met, and the detection result is high in accuracy; the detection sensitivity can reach 200 copies / mL, and the detection time is less than 35 minutes.

Description

Technical Field

[0001] This invention relates to the field of molecular biology detection, specifically to a composition and kit for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. Background Technology

[0002] Gonococci (NG) primarily infect the columnar epithelial cells of the human urogenital tract and can spread further from the urethra and cervix, leading to epididymitis, prostatitis, endometritis, salpingitis, pelvic inflammatory disease, and other conditions. Gonococcal infection can cause urethritis, cervicitis, and other urogenital tract infections. If left untreated, it can lead to serious complications, including infertility and pelvic inflammatory disease.

[0003] Chlamydia trachomatis (CT) is a group of Gram-negative bacteria, classified into 14 serotypes. Infection can cause trachoma, keratoconjunctivitis, genitourinary tract infections, and non-gonococcal urethritis. Delayed treatment may even lead to infertility and miscarriage. Chlamydia trachomatis can also cause neonatal inclusion conjunctivitis and infant pneumonia. Early detection and treatment of Chlamydia trachomatis infection are crucial for preventing complications and transmission to others.

[0004] Ureaplasma urealyticum (UU) is a microorganism that parasitizes the human urogenital tract, with a size between that of viruses and bacteria. Ureaplasma urealyticum is one of the important pathogens of sexually transmitted diseases, causing infections of the vagina, cervix, and other reproductive organs, and is closely related to miscarriage, infertility, and intrauterine growth retardation in fetuses. Timely detection and treatment of Ureaplasma urealyticum infection are crucial for preventing complications and protecting reproductive health.

[0005] Currently, several companies on the market have obtained CFDA registration certificates for detection products of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. Their detection time is all over 2 hours, and they all require the use of extraction methods to purify the nucleic acid of the sample, and the amplification time is relatively long. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a composition and kit for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, thereby solving the problem of excessively long detection times in existing technologies.

[0007] To solve the above-mentioned technical problems, the present invention adopts the following technical solution:

[0008] A composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, comprising primer pairs and corresponding probes; the primer pairs comprising: primer pair 1 for Neisseria gonorrhoeae, the nucleotide sequences of which are shown in SEQ ID NO. 01 and SEQ ID NO. 02; primer pair 2 for Chlamydia trachomatis, the nucleotide sequences of which are shown in SEQ ID NO. 03 and SEQ ID NO. 04; and primer pair 3 for Ureaplasma urealyticum, the nucleotide sequences of which are shown in SEQ ID NO. 05 and SEQ ID NO. 06;

[0009] The probes include: a specific probe 1 against Neisseria gonorrhoeae, the nucleotide sequence of which is shown in SEQ ID NO.07; a specific probe 2 against Chlamydia trachomatis, the nucleotide sequence of which is shown in SEQ ID NO.08; and a specific probe 3 against Ureaplasma urealyticum, the nucleotide sequence of which is shown in SEQ ID NO.09.

[0010] Furthermore, each of the specific probes 1, 2, and 3 is connected to a fluorescent labeling group; the fluorescent labels on the specific probes 1, 2, and 3 produce different fluorescent signals.

[0011] Furthermore, the specific probe 1 is connected to the FAM fluorescent labeling group; the specific probe 2 is connected to the JOE fluorescent labeling group; and the specific probe 3 is connected to the CY5 fluorescent labeling group.

[0012] Furthermore, the primer pair also includes a primer pair for detecting human genome β-globin, the nucleotide sequences of which are shown in SEQ ID NO.10 and SEQ ID NO.11; the probe also includes a specific probe for detecting human genome β-globin, the nucleotide sequence of which is shown in SEQ ID NO.012.

[0013] Furthermore, the concentration of each primer in the primer pair is 0.07–0.08 μM; the concentration of each probe is 0.03–0.05 μM.

[0014] Furthermore, primer pair 1 and specific probe 1 targeting Neisseria gonorrhoeae were synthesized with the porA gene of Neisseria gonorrhoeae as the target region; primer pair 2 and specific probe 2 targeting Chlamydia trachomatis were synthesized with the TrpR gene of Chlamydia trachomatis as the target region; and primer pair 3 and specific probe 3 targeting Ureaplasma urealyticum were synthesized with the ure gene of Ureaplasma urealyticum as the target region.

[0015] A kit for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum includes a PCR reaction system, a positive control, a negative control, and a preservation solution; the PCR reaction system includes the composition described above for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum.

[0016] Furthermore, the PCR reaction system also includes buffer and hot-start DNA polymerase.

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0018] Based on the interactions between multiple primers and probes, and the requirements of probe sequences for specificity, sensitivity, and accuracy, this invention screens compositions and kits capable of simultaneously amplifying and detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. The combination of three sets of specific primer pairs and probes designed in this invention can meet the requirements for detection sensitivity and achieve high accuracy. The kit composed of the specific primer pairs and probes of this invention can achieve a detection sensitivity of 200 copies / mL and a detection time of less than 35 minutes.

[0019] The fluorescent labeling groups attached to the specific probes 1, 2, and 3 of this invention produce different fluorescence signals during the PCR process. A single sample can simultaneously detect Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, completing screening and triage in one step. Attached Figure Description

[0020] Figure 1 This is a graph showing the detection results of the positive control standard in Example 2 of the present invention;

[0021] Figure 2 This is a graph showing the detection results of the negative control standard in Example 2 of the present invention. Detailed Implementation

[0022] The technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Parts not mentioned in the present invention are all prior art.

[0023] A composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum includes primer pairs and corresponding probes; the primer pairs include: primer pair 1 for Neisseria gonorrhoeae, the nucleotide sequences of which are shown in SEQ ID NO. 01 and SEQ ID NO. 02; primer pair 2 for Chlamydia trachomatis, the nucleotide sequences of which are shown in SEQ ID NO. 03 and SEQ ID NO. 04; and primer pair 3 for Ureaplasma urealyticum, the nucleotide sequences of which are shown in SEQ ID NO. 05 and SEQ ID NO. 06.

[0024] The probes include: a specific probe 1 against Neisseria gonorrhoeae, the nucleotide sequence of which is shown in SEQ ID NO.07; a specific probe 2 against Chlamydia trachomatis, the nucleotide sequence of which is shown in SEQ ID NO.08; and a specific probe 3 against Ureaplasma urealyticum, the nucleotide sequence of which is shown in SEQ ID NO.09.

[0025] Based on the interactions between multiple primers and probes, and the requirements of probe sequences for specificity, sensitivity, and accuracy, this invention screens compositions and kits capable of simultaneously amplifying and detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. The combination of three sets of specific primer pairs and probes designed in this invention can meet the requirements for detection sensitivity and achieve high accuracy. The kit composed of the specific primer pairs and probes of this invention can achieve a detection sensitivity of 200 copies / mL and a detection time of less than 35 minutes.

[0026] The specific probes 1, 2, and 3 of this invention are all connected to fluorescent labeling groups; specifically, the fluorescent labeling groups on specific probes 1, 2, and 3 produce different fluorescence signals during the PCR process.

[0027] Furthermore, specific probe 1 is linked to the FAM fluorescent labeling group, specific probe 2 is linked to the JOE fluorescent labeling group, and specific probe 3 is linked to the CY5 fluorescent labeling group.

[0028] Furthermore, the concentration of each primer in primer pair 1, primer pair 2, and primer pair 3 is 0.07–0.08 μM; the concentration of specific probe 1, specific probe 2, and specific probe 3 is 0.03–0.05 μM.

[0029] It is understood that the concentration of each primer and the concentration of the specific probe in the primer pairs of this invention refer to their concentration in the PCR reaction system.

[0030] Furthermore, the primer pairs also include primer pairs for detecting human genome β-globin, the nucleotide sequences of which are shown in SEQ ID NO.10 and SEQ ID NO.11; the probes also include specific probes for detecting human genome β-globin, the nucleotide sequences of which are shown in SEQ ID NO.012.

[0031] The concentration of each primer in the human genome β-globin primer pair for detection in this invention is 0.07–0.08 μM, and the concentration of the human genome β-globin-specific probe for detection is 0.03–0.05 μM.

[0032] This invention uses a specific primer pair and probe designed to detect β-globin in the human genome as an internal standard (IC). The probe has a ROX group attached to its 5′ end and a BHQ group attached to its 3′ end.

[0033] Furthermore, the primer pair 1 and specific probe 1 for Neisseria gonorrhoeae were synthesized with the porA gene of Neisseria gonorrhoeae as the target region; the primer pair 2 and specific probe 2 for Chlamydia trachomatis were synthesized with the TrpR gene of Chlamydia trachomatis as the target region; and the primer pair 3 and specific probe 3 for Ureaplasma urealyticum were synthesized with the ure gene of Ureaplasma urealyticum as the target region.

[0034] The probe of this invention uses a highly specific TaqMan probe, which can bind to the corresponding nucleic acid fragment and undergo hydrolysis under the action of exonuclease activity of hot-start DNA polymerase to generate a fluorescent signal. The real-time amplification curve can be obtained based on the relationship between the fluorescent signal and the amplification cycle number, which can be used for the detection of the corresponding pathogen.

[0035] The present invention also discloses a kit for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, comprising a PCR reaction system, a positive control, a negative control, and a preservation solution; the PCR reaction system comprises the composition described above for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum.

[0036] Furthermore, the PCR reaction system also includes buffer and hot-start DNA polymerase.

[0037] The positive control, negative control, preservation solution, buffer solution, and hot-start DNA polymerase in the kit of this invention are all prepared using conventional techniques in the art, and will not be described in detail here.

[0038] The PCR reaction system of this invention is in lyophilized form. The lyophilization steps are as follows: The PCR reaction system is pre-cooled, sublimation-drying lyophilized, and desorption-drying lyophilized to obtain lyophilized PCR amplification reagent; Pre-cooling stage: The pre-cooling temperature is -50 to -40℃, and the pre-cooling time is 150 to 200 min; Sublimation-drying lyophilization stage: Under a vacuum of 0 to 0.1 mbar, it is lyophilized at -40 to -30℃ for 100 to 150 min, then heated to -30 to -20℃ for 220 to 260 min, and then heated again to -20 to -10℃ for 150 to 200 min; Desorption-drying stage: The temperature is maintained at 4 to 10℃ for 30 to 40 min, and then heated to 10 to 20℃ for 30 to 40 min to obtain lyophilized PCR amplification reagent.

[0039] The PCR reaction system of this invention in lyophilized form can be stored at room temperature for up to 12 months, and the detection sensitivity and precision meet the detection requirements.

[0040] This invention also discloses a method for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, comprising the following steps:

[0041] The sample was placed in the preservation solution of the kit. 60 μL of the preservation solution containing the sample was transferred into a PCR amplification tube containing the PCR reaction system. The tube was then placed on a fluorescent PCR instrument for PCR amplification. The amplification program was as follows: pre-denaturation at 97℃ for 30 seconds; 97℃ for 8 seconds; 57℃ for 15 seconds, 42 cycles (fluorescence collection); and finally, holding at 4–16℃. The fluorescence signal of the corresponding pathogen gene was obtained, and the presence of the corresponding pathogen in the sample was confirmed based on the fluorescence signal.

[0042] Positive and negative controls were placed into PCR amplification tubes containing the PCR reaction system and placed on a fluorescence PCR instrument for PCR amplification. The amplification program was as follows: pre-denaturation at 97℃ for 30 seconds; 97℃ for 8 seconds; 57℃ for 15 seconds, 42 cycles (for fluorescence collection); and finally, holding at 4–16℃ to obtain the corresponding fluorescence signals. The positive control was used to detect whether the kit was functioning properly, and the negative control was used to check for contamination of the kit.

[0043] The preservation solution includes 16–20 mM Tris-HCl, 16–20 mM NaCl, 4–6 mg / mL KCl, 0.1–0.2 v% Tween 20, 0.1–0.2 v% Triton X-100, 0.1–0.2 v% Nonidet P-40, 10–20 mg / mL NaOH, 8–10 mg / mL betaine, 0.04–0.05 mg / mL proteinase K, and 0.8–1 mg / mL lithium dodecyl sulfate.

[0044] Amplification was performed using polymerase chain reaction (PCR), which could be carried out in a PCR reaction system. The buffer components of the PCR reaction system were: 50–70 mM Tris-HCl, 25–45 mM KCl, 2–2.5 mM MgCl2, 0.6–0.8 mM dNTPs, 10–20 mM (NH4)2SO4, 0.01–0.02 v% BSA, 0.02–0.05 v% Tween 20, 0.005–0.01 w% gelatin, 0.01–0.02 v% glycerol, 0.05–0.1 v% Triton-X, 0.1–0.2 v% DMSO, 8–10 mM TMAC, 1.5–3 w% trehalose, and 1–2 w% sucrose.

[0045] The kit provided by this invention has a wide annealing temperature range, and the amplification system can be used on various general-purpose fluorescence PCR instruments and rapid amplification fluorescence PCR instruments. The optimal amplification program is: pre-denaturation at 97°C for 30 seconds; 97°C for 8 seconds, 57°C for 15 seconds, for 42 cycles (fluorescence collection); and finally, hold at 4–16°C.

[0046] This kit can be used for extraction-free sample detection, with no significant difference in results compared to extraction-based detection, greatly shortening the detection time and making it suitable for POCT (Point-of-Care Testing).

[0047] Example 1

[0048] 1. Design of primer pairs and probes for detection systems of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum.

[0049] Primer and probe lengths: Each primer should be 16–30 bases long, and the fluorescent probe should be 20–35 bases long; the GC% of primers and probes should be 40–60%, the primer Tm value should be 50–60℃, and the probe Tm value should be 8–10℃ higher than the primer Tm value; base pairing should be avoided as much as possible at the 3' ends of primers, probes, and between primers and probes; more than 6 consecutive base pairs should be avoided as much as possible at the 5' ends of primers, probes, and between primers and probes; the target fragment length amplified by upstream and downstream primers should be 100–150 bp, and the probe should be located between upstream and downstream primers, as close as possible to the upstream primer; guanine should be avoided at the 5' end of the probe; the selected primers and probes should be designed in conserved regions of the genomes of Neisseria gonorrhoeae, Chlamydia trachomatis, or Ureaplasma urealyticum, respectively, and should be able to specifically detect the target viral nucleic acid while having no cross-reactivity with other species.

[0050] The genomic sequences of Neisseria gonorrhoeae, Chlamydia trachomatis, or Ureaplasma urealyticum were retrieved from GenBank and homology comparisons were performed to identify conserved homologous regions of the NG, CT, or UU genes and the internal standard β-globin gene. Using the Blast tool in the GenBank database, three sets of primers and probes were designed, as shown in Table 1. The NG gene probe was labeled with a fluorescent reporter group (FAM) at the 5′ end and DBQ1 at the 3′ end; the CT gene probe was labeled with a fluorescent reporter group (JOE) at the 5′ end and BHQ1 at the 3′ end; and the UU gene probe was labeled with a fluorescent reporter group (CY5) at the 5′ end and BHQ2 at the 3′ end.

[0051] Table 1. Information on the three candidate primer pairs and probes

[0052]

[0053]

[0054] 2. Screening of primer pairs and probes for the detection system of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum.

[0055] The above primer pairs and probes were used to perform three repeated tests on NG, CT and UU reference samples with 200 copies / mL, and the Ct values ​​and test results were calculated. The test results are shown in Table 2 below.

[0056] Table 2. Comparison of detection sensitivity tests for three candidate primer pairs and probes

[0057]

[0058] Comparison of the detection results of the three primer pairs and probes revealed that the first primer pair and probe could not reliably detect 200 copies / mL of UU reference material, the second primer pair and probe could not detect 200 copies / mL of CT and UU reference materials, and the third primer pair and probe (i.e., the primer pair and probe provided by this invention) could reliably detect 200 copies / mL of NG, CT and UU reference materials. Therefore, the third primer pair and probe is the optimal one, with higher detection sensitivity, and can detect 200 copies / mL of reference material, meeting the requirement of high sensitivity.

[0059] Example 2

[0060] Primer pairs and corresponding probes were designed for Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, respectively. The primer and probe sequences, concentrations, and fluorescent labeling methods are shown in Table 3. Furthermore, this kit uses human genomic β-globin as an internal standard (IC), and its primer and probe information is as follows:

[0061] Primer pair sequence (5′-3′):

[0062] SEQ ID NO.10: GCTTAATTTGCTTCTGACA;

[0063] SEQ ID NO.11: AGTAACGGCAGACTTCTCC;

[0064] Probe sequence (5′-3′):

[0065] SEQ ID NO. 12: ACTAGCAACCTCAAACAGACACC.

[0066] Table 3 Primer pair and probe information

[0067]

[0068] The reagent kit components for preparing detection systems for Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum are as follows:

[0069]

[0070] The PCR amplification system for each 60 μL is as follows: PCR amplification reagent lyophilized, 1 person, and preservation solution containing sample template, 60 μL.

[0071] The positive control contains an 800bp fragment extracted from the corresponding target region, with a concentration of 200,000 copies / mL.

[0072] Example 3

[0073] 1. Amplification of positive control standard

[0074] One lyophilized sample of the PCR reaction system prepared in Example 2 was mixed with 60 μL of positive control. After mixing, the mixture was briefly centrifuged and then placed in a fluorescence PCR instrument (the instrument used in this example was a GN7120 nucleic acid analyzer, GeneDan Biotechnology Co., Ltd.) for PCR amplification.

[0075] PCR amplification program: pre-denaturation at 97℃ for 30 seconds; 97℃ for 8 seconds and 57℃ for 15 seconds, 42 cycles (for fluorescence collection); final hold at 4-16℃.

[0076] After amplification, data analysis was performed using GN7120, and the detection results are as follows: Figure 1 As shown, the horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence signal. The amplification curves of NG, CT, UU, and IC are clear and exhibit a standard S-shaped curve with no significant fluctuation in fluorescence background.

[0077] 2. Amplification of negative control standard

[0078] One lyophilized sample of the PCR reaction system prepared in Example 2 was mixed with 60 μL of negative control. After mixing, the mixture was briefly centrifuged and then placed in a fluorescence PCR instrument (the instrument used in this example was a GN7120 nucleic acid analyzer, GeneDan Biotechnology Co., Ltd.) for PCR amplification.

[0079] PCR amplification program: pre-denaturation at 97℃ for 30 seconds; 97℃ for 8 seconds and 57℃ for 15 seconds, 42 cycles (for fluorescence collection); final hold at 4-16℃.

[0080] After amplification, data analysis was performed using GN7120, and the detection results are as follows: Figure 2 As shown, the horizontal axis represents the cycle number, and the vertical axis represents the relative fluorescence signal value. The IC amplification curve is clear and exhibits a standard S-shaped curve. The fluorescence background shows no significant fluctuations, and no non-specific amplification occurs.

[0081] Example 4

[0082] Comparison of the detection kit of this invention with commercially available kits

[0083] 1. Sample and commercially available reagent kit information

[0084] Twenty cervical swabs and twenty urethral swabs were provided by Nanjing Jiangbei People's Hospital. Two specimens were collected from the same patient; one was placed in the preservation solution of the kit of this invention, and the other was placed in the preservation solution of the control kit.

[0085] The comparison kit was a Chlamydia trachomatis / Ureaplasma urealyticum / Neisseria gonorrhoeae nucleic acid detection kit (PCR-fluorescent probe method) (National Medical Device Registration Certificate No. 20193400148), purchased from Sansure Biotech Inc.

[0086] 2. Sample pretreatment

[0087] Samples collected using cervical or urethral swabs are placed in the preservation solution of their respective kits. The kits of this invention can be directly tested after sampling. For comparison kits, the recommended procedures are as follows: Specific steps and times are shown in Table 4.

[0088] Table 4 Comparison of pretreatment steps between the reagent kit of the present invention and the comparative reagent kit.

[0089]

[0090]

[0091] 3. Amplification Procedure

[0092] The kit of this invention uses a rapid amplification program. The comparison kit was amplified according to the instructions. The specific amplification program and time are shown in Table 5.

[0093] Table 5 Comparison of amplification procedures between the reagent kit of this invention and commercially available reagent kits.

[0094]

[0095] 4. Sample testing

[0096] The PCR reaction solution of the kit of the present invention was prepared according to Example 2 and lyophilized, and then rapidly amplified on a fluorescent PCR detector; the control kit was operated according to its instructions and rapidly amplified on a fluorescent PCR detector. The detection results are shown in Table 6. According to the data statistics, the positive concordance rate of the kit of the present invention was 100%, the negative concordance rate was 100%, and the overall concordance rate was 100% compared with the control kit.

[0097] Table 6 Comparison of positive and negative test results between the kit of the present invention and the comparative kit

[0098]

[0099]

[0100]

[0101] 5. Reference sample testing

[0102] Reference samples for Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum were serially diluted to obtain reference samples of 2000 copies / mL, 1000 copies / mL, 800 copies / mL, 400 copies / mL, and 200 copies / mL. These samples were then tested using the kit of this invention and the comparative kit, with the tests repeated 20 times. The detection rates were then calculated.

[0103] The test results are shown in Table 7. According to the data statistics, the kit of the present invention has a detection sensitivity of up to 200 copies / mL (detection rate ≥95%) compared with the comparative kit, which is higher.

[0104] Table 7 Comparison of test results between the present invention's kit and the comparative kit's reference material.

[0105]

[0106] Example 5

[0107] Performance verification of the detection kit of the present invention

[0108] 1. Precision verification

[0109] Precision was validated using reference samples and borderline positive clinical samples for Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. Precision validation followed the precision testing method in CLSI Guideline EP05-A3, using a "20x2x2" model. Reference samples for Chlamydia trachomatis, Neisseria gonorrhoeae, and Ureaplasma urealyticum were diluted to 500 copies / mL. Precision testing was then performed on a mixed sample of 500 copies / mL of NG, CT, and UU reference samples.

[0110] Two individuals (A and B) conducted tests for 20 days, repeating the process twice daily. The Ct values ​​were statistically analyzed, and the coefficient of variation (CV) was calculated. The results are shown in Table 8. Statistical analysis showed that the CV for each detection target in this invention was less than 5%.

[0111] Table 8 Precision verification of the detection kit of the present invention

[0112]

[0113]

[0114]

[0115]

[0116]

[0117] The precision CV values ​​obtained based on the above data are shown in Table 9:

[0118] Table 9 CV values ​​of the detection kit of the present invention

[0119]

[0120]

[0121] Note: S R Standard deviation, coefficient of variation (CV)

[0122] Based on the above experiments, under the same conditions for 20 days of testing, with two analytical batches per day and each batch measured twice by different people, a total of 80 test results were obtained. The results showed that the detection precision of the kits for Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum was all within 5%.

[0123] 2. Detection sensitivity verification

[0124] Reference standards for Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum were serially diluted to obtain 200 copies / mL. The detection sensitivity of a mixed sample of 200 copies / mL of NG, CT, and UU reference standards was tested. The test was repeated 20 times. The detection results are shown in Table 10. Statistical analysis showed that the detection rate for each target was 100%. The kit of this invention has a detection sensitivity of up to 200 copies / mL.

[0125] Table 10 Sensitivity verification of the detection kit of the present invention

[0126]

[0127] 3. Detection specificity verification

[0128] The samples tested were parasitic microorganisms or sexually transmitted pathogens of the human urinary and reproductive tracts. They were categorized into 19 types of bacteria, including Lactobacillus acidophilus, Staphylococcus epidermidis, Staphylococcus aureus, Enterococcus faecalis, Streptococcus pyogenes, Streptococcus agalactiae, Candida spp., Escherichia coli, Enterococcus, Clostridium difficile, Streptococcus, Klebsiella pneumoniae, Enterobacter, Proteus, Pseudomonas, Bacteroides, Bifidobacterium, Fusarium, and Pallidaceae; 7 types of pathogens, including adenovirus, cytomegalovirus, Epstein-Barr virus, herpes simplex virus 1, herpes simplex virus 2, HPV16, and HPV18; and Candida albicans and Trichomonas vaginalis.

[0129] Using cervical samples from healthy individuals, the cross-reactive pseudoviruses and bacteria were diluted to 10⁻⁶. 5 PFU / mL and 10 6 CFU / mL. Cross-reactivity assessment consists of two aspects: gene sequence alignment and cross-reactivity validation.

[0130] a) Gene sequence alignment

[0131] The whole genome sequences of the cross-reactive samples were obtained from NCBI and clustered with the target sequences of this invention. The detection results are shown in Table 11. Statistical analysis showed that the target sequences in the kit of this invention have no homology with the gene sequences of parasitic microorganisms or sexually transmitted pathogens of the human urinary and reproductive tracts.

[0132] Table 11 Results of gene sequence comparison

[0133]

[0134]

[0135] b) Verification of cross-reactivity

[0136] The test results were obtained using the kit of the present invention and are shown in Table 12. According to the data statistics, there is no cross-interference between common parasitic microorganisms or sexually transmitted pathogens of the human urinary tract and the kit of the present invention.

[0137] Table 12 Cross-reactivity validation results

[0138] Cross-reactive samples Sample concentration Test results Cross-reactive samples Sample concentration Test results Lactobacillus acidophilus <![CDATA[10 6 CFU / mL]]> Negative Pseudomonas <![CDATA[10 6 CFU / mL]]> Negative Staphylococcus epidermidis <![CDATA[10 6 CFU / mL]]> Negative Bacteroides <![CDATA[10 6 CFU / mL]]> Negative Staphylococcus aureus <![CDATA[10 6 CFU / mL]]> Negative Bifidobacteria <![CDATA[10 6 CFU / mL]]> Negative Enterococcus faecalis <![CDATA[10 6 CFU / mL]]> Negative Fusarium <![CDATA[10 6 CFU / mL]]> Negative Streptococcus pyogenes <![CDATA[10 6 CFU / mL]]> Negative Palecoccus <![CDATA[10 6 CFU / mL]]> Negative agalactococcus <![CDATA[10 6 CFU / mL]]> Negative adenovirus <![CDATA[10 5 SMALL / mL]]> Negative Candida <![CDATA[10 6 CFU / mL]]> Negative Cytomegalovirus <![CDATA[10 5 SMALL / mL]]> Negative E. coli <![CDATA[10 6 CFU / mL]]> Negative EB virus <![CDATA[10 5 SMALL / mL]]> Negative Enterococcus <![CDATA[10 6 CFU / mL]]> Negative Herpes simplex virus 1 <![CDATA[10 5 SMALL / mL]]> Negative Clostridium <![CDATA[10 6 CFU / mL]]> Negative Herpes simplex virus 2 <![CDATA[10 5 SMALL / mL]]> Negative Streptococcus <![CDATA[10 6 CFU / mL]]> Negative HPV16 <![CDATA[10 6 copies / reaction]]> Negative Klebsiella pneumoniae <![CDATA[10 6 CFU / mL]]> Negative HPV18 <![CDATA[10 6 copies / reaction]]> Negative Enterobacteriaceae <![CDATA[10 6 CFU / mL]]> Negative Candida albicans - Negative Proteus <![CDATA[10 6 CFU / mL]]> Negative Trichomonas vaginalis <![CDATA[10 3 copies / reaction]]> Negative

[0139] 4. Interference verification

[0140] Endogenous interfering substances included hemoglobin, leukocytes, and mucin; four concentration levels were set for each interfering substance: hemoglobin 160 g / L, 180 g / L, 200 g / L, and 220 g / L; leukocytes 40 / HPF, 45 / HPF, 50 / HPF, and 55 / HPF; and mucins 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, and 1 mg / mL. Using healthy human samples containing different endogenous interfering substances, NG, CT, and UU were diluted to the detection limit (200 copies / mL) and measured 20 times repeatedly.

[0141] Exogenous interfering substances include nonanol ether suppositories, mifepristone tablets, peony root extract vaginal effervescent tablets, compound clotrimazole cream, econazole, Baofukang suppositories, and human lubricants; each interfering substance is prepared at four concentration levels, including nonanol ether suppositories at 6 mg / mL, 8 mg / mL, 10 mg / mL, and 12 mg / mL; mifepristone tablets at 2.0 mg / L, 2.2 mg / L, 2.4 mg / L, and 2.6 mg / L; and 60,000 IU / mL, 80,000 IU / mL, 100,000 IU / mL, and 12 mg / mL. Nistin vaginal effervescent tablets (0000 IU / mL); clotrimazole cream (0.06 g / mL, 0.08 g / mL, 0.1 g / mL, 0.12 g / mL); econazole (0.06 g / mL, 0.08 g / mL, 0.1 g / mL, 0.12 g / mL); bafukang suppositories (0.06 g / mL, 0.08 g / mL, 0.1 g / mL, 0.12 g / mL); and human lubricant (0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 0.6 g / mL). Using healthy human samples containing different exogenous interfering substances, NG, CT, and UU were diluted to the detection limit (200 copies / mL) and measured 20 times repeatedly.

[0142] a) Endogenous interfering substances

[0143] The detection results of endogenous interfering substances using the kit of this invention are shown in Table 13. The presence of 200 g / L hemoglobin, 50 / HPF white blood cells, and 0.9 mg / mL mucin in the sample had no effect on the detection results of this kit.

[0144] Table 13 Test results of endogenous interfering substances

[0145]

[0146] b) Exogenous interfering substances

[0147] The detection results of exogenous interfering substances using the kit of this invention are shown in Table 14. The presence of 10 mg / mL nonoxynol ether suppositories, 2.4 mg / L mifepristone tablets, 100,000 IU / mL nistin vaginal effervescent tablets, 0.1 g / mL compound clotrimazole cream, 0.1 g / mL econazole, 0.1 g / mL Baofukang suppositories, and 0.5 g / mL human lubricant in the sample had no effect on the detection results of this kit.

[0148] Table 14 Test Results of Exogenous Interference Substances

[0149]

[0150]

[0151] 5. Stability Verification

[0152] The PCR amplification reagents in the kit provided by this invention are presented in lyophilized form and can be stored at room temperature, remaining stable for up to 12 months. The kit prepared in Example 2 was stored at 28°C, and kits within one month of their expiration date were used to verify the detection sensitivity and precision.

[0153] Stability verification was performed using reference samples of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. The reference samples of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum were serially diluted to obtain 2000 copies / mL, 200 copies / mL, and so on.

[0154] Precision testing was performed on a mixed sample of NG, CT, and UU reference standards at a concentration of 2000 copies / mL. Two individuals (A and B) conducted the tests for 20 days, repeating twice daily. The Ct values ​​were calculated, and the coefficient of variation (CV) was determined. The results are shown in Table 15. Sensitivity testing was performed on a mixed sample of NG, CT, and UU reference standards at a concentration of 200 copies / mL, with 20 repeated tests. The results are shown in Table 16. Testing criteria: 1) Sensitivity: Detection rate of each target should be ≥95%; 2) Precision: Coefficient of variation (CV) of Ct values ​​for each target should be ≤5%.

[0155] Table 15 Precision test results of kits within one month of their expiration date:

[0156]

[0157] The precision CV values ​​obtained based on the above data are as follows:

[0158]

[0159] Note: S R Standard deviation, coefficient of variation (CV)

[0160] Table 16: Detection sensitivity test results of kits within one month of their expiration date:

[0161]

[0162]

[0163] Stability verification was performed on kits that were one month past their expiration date. The results showed that: sensitivity testing of a mixed sample of 200 copies / mL of NG, CT, and UU reference standards yielded 100% detection rate for each target; precision testing of a mixed sample of 2000 copies / mL of NG, CT, and UU reference standards showed coefficients of variation (CVs) of 1.78%, 1.68%, and 1.68% for the Ct values ​​of the NG, CT, and UU targets, respectively. Both the sensitivity and precision test results met the testing standards, indicating that this kit can be stored at 28°C for 12 months.

[0164] This invention relates to a composition and kit for the detection of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum. These kits feature strong fluorescence signals and high sensitivity. The PCR amplification reagents in the kits are presented in lyophilized form, allowing for storage at room temperature with a shelf life of 12 months. The kits provide highly accurate results, with a detection sensitivity of up to 200 copies / mL. Furthermore, these kits can be used for extraction-free sample detection, showing no significant difference in performance compared to extraction methods, greatly shortening the detection time and making them suitable for point-of-care testing (POCT).

[0165] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended 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 or all of the technical features therein; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, characterized in that, It includes primer pairs and corresponding probes; the primer pairs include: primer pair 1 for Neisseria gonorrhoeae, whose nucleotide sequences are shown in SEQ ID NO. 01 and SEQ ID NO. 02; primer pair 2 for Chlamydia trachomatis, whose nucleotide sequences are shown in SEQ ID NO. 03 and SEQ ID NO. 04; and primer pair 3 for Ureaplasma urealyticum, whose nucleotide sequences are shown in SEQ ID NO. 05 and SEQ ID NO.

06. The probes include: a specific probe 1 against Neisseria gonorrhoeae, the nucleotide sequence of which is shown in SEQ ID NO.07; a specific probe 2 against Chlamydia trachomatis, the nucleotide sequence of which is shown in SEQ ID NO.08; and a specific probe 3 against Ureaplasma urealyticum, the nucleotide sequence of which is shown in SEQ ID NO.

09.

2. The composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum according to claim 1, characterized in that, The specific probes 1, 2, and 3 are all connected to fluorescent labeling groups; the fluorescent labels on the specific probes 1, 2, and 3 produce different fluorescence signals.

3. The composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum according to claim 2, characterized in that, The specific probe 1 is connected to the FAM fluorescent labeling group; the specific probe 2 is connected to the JOE fluorescent labeling group; and the specific probe 3 is connected to the CY5 fluorescent labeling group.

4. The composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum according to claim 1, characterized in that, The primer pair also includes a primer pair for detecting human genome β-globin, the nucleotide sequences of which are shown in SEQ ID NO. 10 and SEQ ID NO. 11; the probe also includes a specific probe for detecting human genome β-globin, the nucleotide sequence of which is shown in SEQ ID NO.

012.

5. A composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum according to any one of claims 1-4, characterized in that, The concentration of each primer in the primer pair is 0.07–0.08 μM; the concentration of each probe is 0.03–0.05 μM.

6. The composition for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum according to claim 1, characterized in that, Primer pair 1 and specific probe 1 targeting Neisseria gonorrhoeae were synthesized with the porA gene of Neisseria gonorrhoeae as the target region; primer pair 2 and specific probe 2 targeting Chlamydia trachomatis were synthesized with the TrpR gene of Chlamydia trachomatis as the target region; primer pair 3 and specific probe 3 targeting Ureaplasma urealyticum were synthesized with the ure gene of Ureaplasma urealyticum as the target region.

7. A kit for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum, characterized in that, It includes a PCR reaction system, a positive control, a negative control, and a preservation solution; the PCR reaction system includes the composition for the detection of Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum as described in any one of claims 1-6.

8. The kit for detecting Neisseria gonorrhoeae, Chlamydia trachomatis, and Ureaplasma urealyticum according to claim 7, characterized in that, The PCR reaction system also includes buffer and hot-start DNA polymerase.

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