A digital PCR detection primer and probe combination for Ureaplasma urealyticum and a kit thereof

Abstract

The present invention discloses a digital PCR detection primer and probe combination for Ureaplasma urealyticum and a kit thereof. The present invention analyzes and compares the genome sequences of 14 different serotypes of Ureaplasma urealyticum. Based on the comparison analysis results, a digital PCR detection primer and probe combination for Ureaplasma urealyticum was designed and optimized, and a corresponding detection kit was obtained. Using the kit described in the present invention, combined with a droplet digital PCR platform, quantitative detection of Ureaplasma urealyticum can be achieved in as fast as 2.5 hours. It has the advantages of high sensitivity, good specificity, and accurate detection results. Its minimum detection limit is 300 copies / mL, and the sample concentration is greater than 9×10 3 When the test results were 100 copies / mL, the difference between the measured value and the theoretical value was less than 15%. In addition, when used for clinical sample testing, the test results of the kit were 100% consistent with the actual results, showing excellent accuracy.

Description

Technical Field

[0001] The present invention belongs to the technical field of pathogen detection and more specifically relates to a digital PCR detection primer and probe combination for Ureaplasma urealyticum and a kit thereof. Background Art

[0002] Ureaplasma urealyticum (UU) is a species in the genus Ureaplasma, order Mycoplasmatales, family Mycoplasmataceae, class Mollicutes. It is a spherical prokaryotic microorganism with no cell wall and few organelles. It is named for its requirement for urea for growth. In 2007, the International Association for Taxonomy of Bacteriology defined two new species within UU: Ureaplasma parvum (UPA biogroup I) and Ureaplasma urealyticum (UUR biogroup II). UPA biogroup I includes four serotypes: 1, 3, 6, and 14, while UUR biogroup II includes ten serotypes: 2, 4, 5, 7, 8, 9, 10, 11, 12, and 13.

[0003] UU is a common conditional pathogen of the human urogenital tract, usually living on the surface of the urogenital tract. Infected people often have no obvious clinical symptoms and lack more specific clinical manifestations. When the body's immunity is reduced, or it is subjected to invasive operations or invaded by other pathogens, UU will break through the mucosal layer, causing non-gonococcal urethritis and a variety of other urogenital tract diseases, such as prostatitis and epididymitis. It can also cause infertility, spontaneous abortion, and lead to birth canal infection during delivery. The amount of UU carried is an important indicator to measure its pathogenicity. Therefore, compared with qualitative detection of UU, accurate quantitative detection is more conducive to the prevention and treatment of UU-related diseases.

[0004] At present, there are two methods for quantitative detection of UU: real-time fluorescence quantitative PCR and digital PCR (ddPCR). Among them, real-time fluorescence quantitative PCR relies on a standard curve, has poor discrimination of target gene molecules with low copy numbers, and is easily limited in detection sensitivity, accuracy, and resolution. Digital PCR is a nucleic acid absolute quantification technology that has developed rapidly in recent years. It solves the deficiency that real-time fluorescence quantitative PCR needs to rely on a standard curve for quantitative analysis. Through the endpoint detection method, the number of target DNA molecules can be directly calculated to achieve absolute quantification of "single molecule template amplification". Although there are reports on methods and kits for quantitative detection of UU based on digital PCR, most of them are single-plex detection systems, and the reports do not clearly disclose the difference between the quantitative detection results of the samples and the actual concentration of the samples. The linear analysis results provided cannot directly indicate that the quantitative accuracy is good. At the same time, clinical samples have not been tested, so the accuracy of identification of positive clinical samples is unknown. Summary of the Invention

[0005] In view of the deficiencies in the above-mentioned prior art, the present invention provides a digital PCR detection primer and probe combination for Ureaplasma urealyticum and a kit thereof, which realizes sensitive, specific and accurate detection of Ureaplasma urealyticum.

[0006] The first object of the present invention is to provide a digital PCR detection primer and probe combination for Ureaplasma urealyticum.

[0007] The second object of the present invention is to provide an application of the digital PCR detection primer and probe combination in the preparation of a detection product for Ureaplasma urealyticum.

[0008] The third object of the present invention is to provide a detection kit for Ureaplasma urealyticum.

[0009] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0010] This study analyzed and compared the genome sequences of different Ureaplasma urealyticum strains (including 14 different serotypes). Based on the comparison results, a digital PCR primer and probe combination for Ureaplasma urealyticum detection was designed and optimized. Based on droplet digital PCR technology, this digital PCR primer and probe combination can achieve accurate and quantitative detection of Ureaplasma urealyticum.

[0011] Specifically, a droplet reader measures the fluorescence level of each droplet to determine its positive or negative status. By tallying the positive and negative status of all droplets, the presence of U. urealyticum nucleic acid in the sample can be determined, enabling qualitative detection of U. urealyticum. Based on the statistical results of positive and negative droplets and combined with statistical formulas, the copy number of U. urealyticum nucleic acid in the sample can be further calculated, enabling quantitative detection of U. urealyticum.

[0012] The present invention provides a digital PCR detection primer and probe combination for Ureaplasma urealyticum, comprising a digital PCR primer pair and a probe for detecting Ureaplasma urealyticum; the nucleotide sequence of the digital PCR primer pair is shown in SEQ ID NOs. 1 and 2, and the nucleotide sequence of the probe is shown in SEQ ID NO. 5; the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

[0013] Specifically, the Ureaplasma urealyticum includes 14 different serotypes thereof.

[0014] As one embodiment, the fluorescent group labeled at the 5' end of the probe for detecting Ureaplasma urealyticum is FAM, and the quenching group labeled at the 3' end is MGB.

[0015] To avoid the problem of false negatives, the digital PCR detection primer and probe combination for Ureaplasma urealyticum described in the present invention also includes a digital PCR primer pair and probe for detecting an internal standard gene; the nucleotide sequence of the digital PCR primer pair is shown in SEQ ID NOs. 3 to 4, and the nucleotide sequence of the probe is shown in SEQ ID NO. 6; the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; the fluorescent group is different from the one labeled on the probe for detecting Ureaplasma urealyticum.

[0016] As one embodiment, the fluorescent group labeled on the 5' end of the probe for detecting the internal standard gene is VIC, and the quenching group labeled on the 3' end is MGB.

[0017] The digital PCR detection primer and probe combination of the present invention can achieve quantitative detection of Ureaplasma urealyticum. Therefore, the present invention claims protection for the use of the digital PCR detection primer and probe combination in the preparation of a detection product for Ureaplasma urealyticum.

[0018] Optionally, the detection product includes a qualitative or quantitative detection product.

[0019] The invention also provides a Ureaplasma urealyticum detection kit.

[0020] Specifically, the kit contains a primer-probe premix and reagents required for a digital PCR reaction; the primer-probe premix contains the digital PCR detection primer and probe combination of the present invention.

[0021] Specifically, the reagents required for the digital PCR reaction are reagents required for a droplet digital PCR reaction.

[0022] Specifically, the reagent used in the preparation of the primer-probe premix solution of the present invention is Tris-hydrochloric acid buffer.

[0023] The kit of the present invention also contains a positive quality control product and a negative quality control product.

[0024] Specifically, the positive quality control product contains the nucleic acid fragment (gene sequence) of the detected Ureaplasma urealyticum and an internal standard gene sequence.

[0025] Optionally, the positive quality control product is a recombinant plasmid or pseudovirus containing the nucleic acid fragment of the detected Ureaplasma urealyticum and an internal standard gene sequence.

[0026] Specifically, the negative control product contains an internal standard gene sequence.

[0027] Optionally, the negative control product is a recombinant plasmid or pseudovirus containing only the internal standard gene sequence.

[0028] The present invention also provides a method for quantitatively detecting Ureaplasma urealyticum using the kit of the present invention, comprising the following steps:

[0029] S1. Extracting nucleic acid from the sample to be tested;

[0030] S2. Prepare a digital PCR reaction system using the sample nucleic acid obtained in S1 as a template;

[0031] S3. Digital PCR detection.

[0032] Optionally, the sample is urogenital secretions.

[0033] Specifically, the reagents used to prepare the reaction system include primer probe premix and droplet digital PCR premix (ddPCR) suitable for DNA samples. TM Supermix for Probes (no dUTP), Bio-rad).

[0034] Specifically, the reaction system (single person) is: 6 μL primer probe premix, 11 μL ddPCR TM Supermix for Probes (no dUTP) and 5 μL sample nucleic acid (or positive control / negative control).

[0035] Specifically, the final concentration range of the upstream and downstream primers of the digital PCR primer pair for detecting Ureaplasma urealyticum and the internal standard gene in the reaction system is 0.3-0.6 μmol / L, and the final concentration range of the probe for detecting Ureaplasma urealyticum and the internal standard gene is 0.15-0.35 μmol / L.

[0036] When the final concentration of the upstream and downstream primers of the digital PCR primer pair for detecting Ureaplasma urealyticum and the internal standard gene in the reaction system is 0.45 μmol / L, and the final concentration of the probe for detecting Ureaplasma urealyticum and the internal standard gene is 0.225 μmol / L, the detection effect is relatively best.

[0037] Specifically, the cycling conditions were: 95° C. for 10 min; 94° C. for 30 s, 55° C. for 1 min 30 s, 40 cycles; 98° C. for 10 min; and storage at 4° C. The FAM channel was used to detect Ureaplasma urealyticum nucleic acid, and the VIC channel was used to detect the internal standard.

[0038] Specifically, the test kit of the present invention is used to determine the effectiveness of the test standard: in each test, only the reaction wells with a droplet count of ≥10,000 are valid reaction wells. The valid reaction wells should include a negative control group and a positive control group. When the positive control group test result is positive, the negative control group test result is negative, and the concentration test result of the positive control group is 10 5The test results are valid when the value is ±10% copies / mL.

[0039] The present invention has the following beneficial effects:

[0040] This study analyzed and compared the genome sequences of 14 different serotypes of Ureaplasma urealyticum. Based on the results of this comparison, a digital PCR primer and probe combination for Ureaplasma urealyticum detection was designed and optimized, along with a corresponding detection kit. Using this kit, combined with a droplet digital PCR platform, quantitative detection of Ureaplasma urealyticum can be achieved in as little as 2.5 hours, demonstrating high sensitivity, good specificity, and accurate results.

[0041] The minimum detection limit of the kit of the present invention is 300 copies / mL. 3 When the number of copies / mL was 100, the difference between the measured value and the theoretical value was less than 15%, indicating that the quantitative detection results were accurate. Furthermore, when used for clinical sample testing, the digital PCR detection primer and probe combination or kit of the present invention achieved 100% consistency with the actual results, demonstrating excellent accuracy and the ability to be used for the quantitative detection of Ureaplasma urealyticum, which is of great significance for the prevention and treatment of Ureaplasma urealyticum-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The digital PCR detection results of the three primer and probe sets shown in Table 1 are shown.

[0043] Figure 2 This is the test result of the kit of the present invention on the negative quality control product of Ureaplasma urealyticum UU; the upper figure in the figure corresponds to the test result of the FAM channel, and the lower figure corresponds to the test result of the VIC channel.

[0044] Figure 3 This is the test result of the kit of the present invention on the positive quality control product of Ureaplasma urealyticum UU; the upper figure in the figure corresponds to the test result of the FAM channel, and the lower figure corresponds to the test result of the VIC channel.

[0045] Figure 4 The kit of the present invention has a concentration of 9×10 6 Digital PCR detection results of inactivated culture of Ureaplasma urealyticum at 100 copies / mL.

[0046] Figure 5 The kit of the present invention has a concentration of 9×10 5 Digital PCR detection results of inactivated culture of Ureaplasma urealyticum at 100 copies / mL.

[0047] Figure 6 The kit of the present invention has a concentration of 9×104 Digital PCR detection results of inactivated culture of Ureaplasma urealyticum at 100 copies / mL.

[0048] Figure 7 The kit of the present invention has a concentration of 9×10 3 Digital PCR detection results of inactivated culture of Ureaplasma urealyticum at 100 copies / mL.

[0049] Figure 8 This is the digital PCR test result of the kit of the present invention on the inactivated culture of Ureaplasma urealyticum with a concentration of 300 copies / mL.

[0050] Figure 9 This is the digital PCR test result of the kit of the present invention on the inactivated culture of Ureaplasma urealyticum with a concentration of 90 copies / mL.

[0051] Figure 10 The upper figure in the figure corresponds to the detection results of the FAM channel, and the lower figure corresponds to the detection results of the VIC channel.

[0052] Figure 11 This is the detection status of the enterprise reference products corresponding to N09~N16; the upper figure corresponds to the detection results of the FAM channel, and the lower figure corresponds to the detection results of the VIC channel.

[0053] Figure 12 The upper figure in the figure corresponds to the detection results of the FAM channel, and the lower figure corresponds to the detection results of the VIC channel. DETAILED DESCRIPTION

[0054] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0055] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0056] Example 1 Design of primer and probe combinations for digital PCR detection of Ureaplasma urealyticum

[0057] The present invention retrieved and downloaded the genome sequences of Ureaplasma urealyticum (including 14 different serotypes) from different sources from the NCBI database and performed a comparative analysis on them. Based on the comparative analysis results, the present invention designed several groups of digital PCR detection primers and probe combinations for different Ureaplasma urealyticum. By synthesizing the designed primer and probe groups and performing digital PCR detection, it was found that the detection effect of the designed primer and probe groups was not ideal, individual primers and probe groups were not available, and most primer and probe groups had the problem of too high fluorescence background of negative droplets. On this basis, the present invention obtained a group of digital PCR detection primers and probe combinations for Ureaplasma urealyticum with a lower fluorescence background of negative droplets through design, screening and optimization, and its nucleotide sequence is shown in Group 3 in Table 1. The other two groups in Table 1 are primer and probe groups eliminated during the screening process of the present invention, as a comparison.

[0058] Table 1 Nucleotide sequences of primers and probes for digital PCR detection of Ureaplasma urealyticum

[0059]

[0060] The probes described in Table 1 are all Taqman probes, with the 5' end of the probes labeled with a FAM fluorescent group and the 3' end of the probes labeled with an MGB quenching group.

[0061] After synthesizing the primer and probe sets shown in Table 1, a fixed-value inactivated culture of Ureaplasma urealyticum was used as the initial sample. A negative urogenital secretion sample was used to dilute the inactivated culture of Ureaplasma urealyticum to a concentration of 12,000 copies / mL, which served as the test sample. After extracting the nucleic acid from the test sample, digital PCR was performed using the combined primer and probe sets. Except for the different primer and probe sets used, all other conditions were identical. The detection process is as follows:

[0062] Prepare the PCR reaction system. Take out the primers and probes to be screened, as well as the ddPCR premix (ddPCR TM Supermix for Probes (no dUTP) was prepared and mixed evenly using a vortex shaker. After centrifugation for 10 seconds, the PCR reaction system was prepared according to Table 2 and aliquoted into PCR reaction tubes.

[0063] Table 2 Ureaplasma urealyticum nucleic acid PCR detection system (single reaction system)

[0064]

[0065]

[0066] Mix the reaction system with the test sample added using a vortex oscillator. Remove a droplet preparation chip and transfer a column (8) of reaction systems to the sample well of the chip. Add 70 μL of droplet preparation oil (Bio-Ra Droplet Generator oil for probes) to the droplet preparation oil well. Transfer the sample to a droplet preparation instrument (Bio-Ra QX200 Droplet Generator) for droplet preparation. Once the instrument reports that droplet preparation is complete, carefully transfer the prepared droplets to a dedicated 96-well plate using a pipette. Discard the droplet preparation chip and use another droplet preparation chip to process the next column of reaction systems until all test reaction systems are processed. Seal the 96-well plate using an aluminum mold on a heat sealer.

[0067] Transfer the sealed 96-well plate to the qualitative PCR and set the reaction conditions as shown in Table 3:

[0068] Table 3 PCR reaction conditions

[0069]

[0070] After the PCR reaction is complete, transfer the 96-well plate to the microplate reader. Set the microplate reading parameters according to the instructions, select the FAM channel for detecting U. ureaplasma urealyticum nucleic acid, and start the microplate reader. Once the microplate reading is complete, the results are automatically saved. Click Analyze to enter the results analysis interface. First, click "Event" to view the total number of droplets in each reaction well. If the number of droplets in a reaction well is less than 10,000, it is considered an invalid reaction well. Next, check whether the positive and negative quality control samples for this experiment are included in the valid reaction wells. If so, the experiment is valid. If the experiment is valid, click "1D Amplitude" to view the fluorescence scatter plot of Ch1. After drawing the threshold line, click "Concentration" to view the concentration of Ch1 in copies / μL. Calculate the concentration of the original sample based on the sample dilution factor.

[0071] The present invention is to detect the primer and probe groups when each reaction well is a valid well. The digital PCR detection results of the three sets of primers and probe groups shown in Table 1 are as follows Figure 1 As shown. Figure 1 It can be seen that primer and probe set 1 did not amplify, indicating that this primer and probe set is unusable; the result of primer and probe set 2 was 14500 copies / mL, and the fluorescence background of the negative droplet was too high, resulting in poor effect; the result of primer and probe set 3 was 12300 copies / mL, and the fluorescence background of the negative droplet was low, resulting in relatively optimal detection effect.

[0072] Example 2 A Ureaplasma urealyticum qualitative and quantitative detection kit

[0073] Based on Example 1, the present invention constructs a Ureaplasma urealyticum qualitative and quantitative detection kit, and the components contained in the kit are shown in Table 4.

[0074] Table 4 Components of the Ureaplasma urealyticum qualitative and quantitative detection kit

[0075]

[0076] The specific primer and probe combination is further optimized on the basis of the primer and probe combination (group 3) described in Example 1. On the basis of group 3, in order to avoid the problem of false negative in detection, the present invention also uses gene GAPDH as the internal standard gene, and designs an internal standard detection primer and probe combination that can be combined with group 3. The specific primer and probe combination finally obtained is specifically shown in Table 5. The nucleotide sequence of the digital PCR detection primer and probe combination of Ureaplasma urealyticum finally optimized by the present invention contains degenerate bases, as shown in the underlined portion in Table 5. Wherein, W corresponds to A / T; Y corresponds to C / T; R corresponds to A / G.

[0077] Table 5 Specific primer and probe combinations

[0078]

[0079] Note: The 5' end of the UU detection probe is labeled with a FAM fluorescent group, and the 3' end is labeled with an MGB quencher group; the 5' end of the internal standard detection probe is labeled with a VIC fluorescent group, and the 3' end is labeled with an MGB quencher group.

[0080] When the detection performance of the kit was subsequently tested, the ddPCR premix was used. TM Supermix for Probes (no dUTP) is a droplet digital PCR premix suitable for DNA samples provided by Bio-rad, with the product number 1863024.

[0081] The steps of using the kit of the present invention to detect the sample to be tested are as follows:

[0082] S1. Prepare PCR reaction system

[0083] Take out the primer probe premix and ddPCR premix from the kit. TM Supermix for Probes (no dUTP) was prepared and mixed evenly using a vortex shaker. After centrifugation for 10 seconds, the PCR reaction system was prepared according to Table 6 and aliquoted into PCR reaction tubes.

[0084] Table 6 Ureaplasma urealyticum nucleic acid PCR detection system (single reaction system)

[0085]

[0086] In the reaction system of the present invention, the final concentration range of the upstream and downstream primers for UU detection and internal standard detection is 0.3-0.6 μmol / L, and the final concentration range of the probes for UU detection and internal standard detection is 0.15-0.35 μmol / L.

[0087] The detection effect was relatively best when the final concentration of the upstream and downstream primers for UU detection and internal standard detection was 0.45 μmol / L and the final concentration of the probe for UU detection and internal standard detection was 0.225 μmol / L.

[0088] S2. Sample addition

[0089] Use the nucleic acid extraction or purification reagents produced by Daan Gene Co., Ltd. (Guangzhou Medical Equipment No. 20170583) to extract and purify the nucleic acids of the collected samples (such as urogenital tract samples). Add 5 μL of the extracted nucleic acid of the sample to be tested, the negative control product, and the positive control product to the above-mentioned PCR reaction tubes, cover the tubes tightly, centrifuge for 15 seconds, and then transfer to the droplet preparation area.

[0090] S3. Microdroplet Preparation

[0091] The reaction system with the added sample was mixed using a vortex oscillator; a droplet preparation chip was taken out, and a column (8) of reaction systems were transferred to the sample well of the chip. 70 μL of droplet preparation oil was added to the droplet preparation oil well, and the system was placed into the droplet preparation instrument for droplet preparation; after the instrument reported that the droplet preparation was complete, a pipette was used to carefully transfer the prepared droplets to a dedicated 96-well plate, the droplet preparation chip was discarded, and another droplet preparation chip was taken to process the next column of reaction systems until all reaction systems to be tested were processed; the 96-well plate was then sealed using an aluminum mold on a heat sealer.

[0092] S4. PCR Amplification

[0093] Transfer the sealed 96-well plate to the qualitative PCR and set the reaction conditions as shown in Table 3.

[0094] S5. Droplet Reading and Validity Analysis

[0095] After PCR, transfer the 96-well plate to the droplet reader and set the droplet reading parameters according to the droplet reader manual. Pay attention to the selection of fluorescence channels: select the FAM channel to detect Ureaplasma urealyticum nucleic acid and select the VIC channel to detect the internal standard. After the settings are completed, start the droplet reading.

[0096] After the droplet reading is completed, the results are automatically saved. Click Analyze to enter the result analysis interface. First, click "Event" to view the total number of droplets in each reaction well. If the number of droplets in a reaction well is less than 10,000, it is considered an invalid reaction well. Then, check whether the valid reaction wells contain the positive and negative quality control products for this test. If so, the test is valid. If the test is valid, click "1D Amplitude" to view the fluorescence scatter plot of Ch1. After drawing the threshold line, click "CoNentration" to view the concentration of Ch1 in copies / μL. Combined with the sample dilution factor, the concentration of the original sample can be calculated.

[0097] The present invention uses the kit to respectively detect the negative quality control product and the positive quality control product (1.0×10 5 The test results of the kit of the present invention on the negative quality control product of Ureaplasma urealyticum UU are as follows: Figure 2 The test results of the kit of the present invention for the positive quality control product of Ureaplasma urealyticum UU are shown as follows. Figure 3 As shown. Figure 2 and Figure 3 It can be seen that the kit can accurately detect Ureaplasma urealyticum.

[0098] Example 3: Sample detection range and sensitivity test of the kit

[0099] The inactivated culture of Ureaplasma urealyticum after determination was used as the initial sample, and the negative urogenital tract secretion sample was used for gradient dilution, and the sample was diluted to 9×10 6 , 9×10 5 , 9×10 4 , 9×10 3 The test was performed at 300, 90 copies / mL, 300, and 90 copies / mL concentrations. After extracting nucleic acids from samples and quality control products, they were detected using the method described in Example 2. The kit of the present invention tested samples at different concentrations (9×10 6 , 9×10 5 , 9×10 4 , 9×10 3 , 300, 90 copies / mL) were tested as follows: Figures 4 to 9 The quantitative detection results (measured values) and their differences with the theoretical values ​​are shown in Table 7.

[0100] Table 7 Sensitivity and quantitative accuracy test results

[0101]

[0102]

[0103] The results shown in Table 7 show that the detection limit of the kit for Ureaplasma urealyticum is 300 copies / mL, and the kit has high sensitivity. 3 When the quantitative detection result of the kit of the present invention is less than 15% of the theoretical value, the quantitative result is highly accurate. In practical applications, the quantitative limit (9×10 3 The detection requirements can be met if the difference between the quantitative detection result of the sample with a concentration of more than 100 copies / mL and the actual concentration is less than 15%.

[0104] Example 4 Specificity detection of the kit

[0105] The present invention selected reference products from companies for 18 pathogens, including microorganisms that parasitize the human urinary and reproductive tracts, sexually transmitted pathogens, and pathogens that are likely to cause the same or similar clinical symptoms as Ureaplasma urealyticum, and performed specific detection on the kit described in Example 2. Specifically, the 18 pathogens were Neisseria gonorrhoeae, Chlamydia trachomatis, Mycoplasma hominis, Mycoplasma genitalium, human papillomavirus type 16, human papillomavirus type 18, human papillomavirus type 45, human papillomavirus type 31, herpes simplex virus type 1, herpes simplex virus type 2, cytomegalovirus, Epstein-Barr virus, group B Streptococcus, Staphylococcus epidermidis, Staphylococcus aureus, Escherichia coli, Candida albicans, and Trichomonas vaginalis.

[0106] The reference products of the above 18 pathogens were used as templates (the template concentration was 10 5 copies / mL), and tested it using the method described in Example 2. The results are as follows Figures 10-12 And as shown in Table 8. Among them, Figure 10 The detection results of the reference products of the corresponding enterprises from N01 to N08 are as follows: Figure 11 The detection results of the reference products of the corresponding enterprises from N09 to N16 are as follows: Figure 12 This is the detection status of the corresponding enterprise reference products for N17 and N18.

[0107] Table 8 Test results of 18 pathogens enterprise reference products

[0108]

[0109]

[0110] Depend on Figures 10-12 As shown in Table 8, the kit of the present invention has no cross-reaction to the above 18 pathogens, indicating that the specific detection of the kit of the present invention meets the requirements.

[0111] Example 6 Clinical application detection of the kit

[0112] Twenty clinically positive and 20 negative samples for UU were selected (these positive and negative samples were tested and identified using a commercially available third-party test kit). After nucleic acid extraction, the samples were labeled to ensure correct labeling and stored at -80°C. The nucleic acids extracted from the 20 clinically positive and 20 negative UU samples were used as templates for testing using the kit and method described in Example 2. The results are shown in Table 9.

[0113] Table 9 Clinical sample test results

[0114]

[0115]

[0116]

[0117] As shown in Table 9, the test results of the kit of the present invention on clinical samples are consistent with the actual results, indicating that the kit of the present invention can accurately determine the negative / positive status of the tested clinical samples.

[0118] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A digital PCR detection primer and probe combination for Ureaplasma urealyticum, characterized in that: It comprises a digital PCR primer pair and a probe for detecting Ureaplasma urealyticum; the nucleotide sequence of the digital PCR primer pair is shown in SEQ ID NO.1-2, and the nucleotide sequence of the probe is shown in SEQ ID NO.5; the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group.

2. The digital PCR detection primer and probe combination according to claim 1, characterized in that: The fluorescent group labeled at the 5' end of the probe used to detect Ureaplasma urealyticum is FAM, and the quenching group labeled at the 3' end is MGB.

3. The digital PCR detection primer and probe combination according to claim 1, characterized in that: It also includes a digital PCR primer pair and probe for detecting the internal standard gene; the nucleotide sequence of the digital PCR primer pair is shown in SEQ ID NO.3-4, and the nucleotide sequence of the probe is shown in SEQ ID NO.6; the 5' end of the probe is labeled with a fluorescent group, and the 3' end is labeled with a quenching group; the fluorescent group is different from that labeled on the probe used to detect Ureaplasma urealyticum.

4. The digital PCR detection primer and probe combination according to claim 3, characterized in that: The 5' end of the probe used to detect the internal standard gene is labeled with a fluorescent group VIC, and the 3' end is labeled with a quencher group MGB.

5. Use of the digital PCR detection primer and probe combination according to any one of claims 1 to 4 in preparing a detection product for Ureaplasma urealyticum.

6. A detection kit for Ureaplasma urealyticum, characterized in that: Contains a primer-probe premix and reagents required for digital PCR reaction; the primer-probe premix contains the digital PCR detection primer and probe combination according to any one of claims 1 to 4.

7. The kit according to claim 6, characterized in that The reagent used to prepare the primer probe premix is ​​Tris-hydrochloric acid buffer.

8. The kit according to claim 6, characterized in that The kit also contains a positive quality control product and a negative quality control product.

9. The kit according to claim 8, characterized in that The positive quality control product contains the nucleic acid fragment of the detected Ureaplasma urealyticum and the internal standard gene sequence.

10. The kit according to claim 8, characterized in that The negative quality control product contains an internal standard gene sequence.

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