Primer probe composition, kit and method for detecting 16 pathogenic microorganisms
Through the primer-probe combination and the integrated nucleic acid extraction and detection device, the problem of difficulty in rapidly detecting multiple pathogenic microorganisms in the existing technology has been solved, and high-sensitivity simultaneous detection of 16 pathogenic microorganisms has been achieved, shortening the detection time.
Patent Information
- Application Number
- CN202510982745.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-07-16
- Publication Date
- 2025-09-19
AI Technical Summary
Existing technologies make it difficult to quickly and sensitively detect a variety of pathogenic microorganisms, and existing microfluidic chips are difficult to produce on a large scale.
Provided is a primer-probe combination and kit, combined with an integrated nucleic acid extraction and detection device, using an optimized lysis solution and fluorescent PCR amplification program to achieve simultaneous detection of 16 pathogenic microorganisms.
It achieves high-sensitivity and rapid detection of multiple pathogenic microorganisms, shortening the detection time to 60 minutes, making it suitable for rapid screening of people with low immunity.
Smart Images
Figure CN120666062A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of molecular biology, and in particular relates to a primer-probe combination, a kit and a method for detecting 16 kinds of pathogenic microorganisms. Background Art
[0002] Pathogens include bacteria, viruses, parasites, etc., which are widely present in nature. Some parasitize the human body, and some exist in nature. Pathogens can be transmitted through air, water, food, contact and other channels. They can cause tuberculosis, cholera, plague, and various infections. They are particularly threatening to people with weakened immunity, such as children, the elderly, and patients with chronic diseases.
[0003] Currently, pathogen detection primarily relies on the following technologies: 1. Traditional culture methods: These methods are time-consuming (24-72 hours), have low sensitivity, and cannot meet the needs of rapid screening. 2. PCR technology: While highly sensitive, they require specialized equipment and personnel and cannot simultaneously detect multiple indicators. 3. Colloidal gold test strips: These methods can only detect a single pathogen, have low throughput, and cannot handle complex infection scenarios.
[0004] Existing microfluidic chips are limited by material complexity and integration process, making it difficult to achieve large-scale production.
[0005] In view of this, it is very necessary to provide a technical solution with fast detection speed, high sensitivity, and the ability to effectively detect multiple pathogenic microorganisms at the same time. Summary of the Invention
[0006] To address at least one of the above-mentioned issues, the present invention provides a primer-probe combination, kit, and method for detecting 16 pathogenic microorganisms. The primer combination screened in this application, combined with integrated extraction and detection using a cartridge, allows for the detection of 16 pathogenic microorganisms, achieving high sensitivity, good extraction efficiency, and reduced detection time.
[0007] In order to achieve the above object, the present invention adopts the following technical means: The first aspect of the present invention provides a primer-probe combination for detecting 16 pathogenic microorganisms, the primer-probe combination comprising: The forward primer for targeted amplification of total colonies is shown in SEQ ID NO.1, the reverse primer is shown in SEQ ID NO.2, and the probe for detecting total colonies is shown in SEQ ID NO.3; The forward primer for targeted amplification of total Vibrio is shown in SEQ ID NO.4, the reverse primer is shown in SEQ ID NO.5, and the probe for detecting total Vibrio is shown in SEQ ID NO.6; The forward primer for targeted amplification of Helicobacter pylori is shown in SEQ ID NO.7, the reverse primer is shown in SEQ ID NO.8, and the probe for detecting Helicobacter pylori is shown in SEQ ID NO.9; The forward primer for targeted amplification of Aeromonas hydrophila is shown in SEQ ID NO.10, the reverse primer is shown in SEQ ID NO.11, and the probe for detecting Helicobacter pylori is shown in SEQ ID NO.12; The forward primer for targeted amplification of Mycobacterium tuberculosis is shown in SEQ ID NO. 13, the reverse primer is shown in SEQ ID NO. 14, and the probe for detecting Mycobacterium tuberculosis is shown in SEQ ID NO. 15; The forward primer for targeted amplification of Vibrio cholerae is shown in SEQ ID NO.16, the reverse primer is shown in SEQ ID NO.17, and the probe for detecting Vibrio cholerae is shown in SEQ ID NO.18; The forward primer for targeted amplification of Yersinia pestis is shown in SEQ ID NO. 19, the reverse primer is shown in SEQ ID NO. 20, and the probe for detecting Yersinia pestis is shown in SEQ ID NO. 21; The forward primer for targeted amplification of Pseudomonas aeruginosa is shown in SEQ ID NO. 22, the reverse primer is shown in SEQ ID NO. 23, and the probe for detecting Pseudomonas aeruginosa is shown in SEQ ID NO. 24; The forward primer for targeted amplification of Staphylococcus aureus is shown in SEQ ID NO. 25, the reverse primer is shown in SEQ ID NO. 26, and the probe for detecting Staphylococcus aureus is shown in SEQ ID NO. 27; The forward primer for targeted amplification of Toxoplasma gondii is shown in SEQ ID NO. 28, the reverse primer is shown in SEQ ID NO. 29, and the probe for detecting Toxoplasma gondii is shown in SEQ ID NO. 30; The forward primer for targeted amplification of Plasmodium is shown in SEQ ID NO.31, the reverse primer is shown in SEQ ID NO.32, and the probe for detecting Plasmodium is shown in SEQ ID NO.33; The forward primer for targeting mycoplasma is shown in SEQ ID NO. 34, the reverse primer is shown in SEQ ID NO. 35, and the probe for detecting Plasmodium is shown in SEQ ID NO. 36; The forward primer for targeted amplification of Chlamydia is shown in SEQ ID NO. 37, the reverse primer is shown in SEQ ID NO. 38, and the probe for detecting Chlamydia is shown in SEQ ID NO. 39; The forward primer for targeted amplification of Rickettsia is shown in SEQ ID NO.40, the reverse primer is shown in SEQ ID NO.41, and the probe for detecting Rickettsia is shown in SEQ ID NO.42; The forward primer for targeted amplification of the novel coronavirus is shown in SEQ ID NO. 43, the reverse primer is shown in SEQ ID NO. 44, and the probe for detecting the novel coronavirus is shown in SEQ ID NO. 45; The forward primer for targeted amplification of HIV is shown in SEQ ID NO.46, the reverse primer is shown in SEQ ID NO.47, and the probe for detecting HIV is shown in SEQ ID NO.48.
[0008] In some embodiments of the present invention, the primer-probe composition is divided into four groups: total colonies, total Vibrio, Helicobacter pylori, and Aeromonas hydrophila are one group; Mycobacterium tuberculosis, Vibrio cholerae, Yersinia pestis, and Pseudomonas aeruginosa are one group; Staphylococcus aureus, Mycoplasma, Chlamydia, and Rickettsia are one group; Toxoplasma, Plasmodium, new coronavirus, and HIV are one group.
[0009] In some embodiments of the present invention, in the primer-probe composition, the 5' end of the probe is connected to a fluorescent group, and the 3' end is connected to a quenching group. The fluorescent group is selected from one of FAM, HEX, CY5, and ROX. The fluorescent labels used on the probes of the same group of pathogenic microorganisms are different.
[0010] The second aspect of the present invention provides the use of the primer-probe combination described in the first aspect in the preparation of products for detecting 16 types of pathogenic microorganisms.
[0011] The third aspect of the present invention provides a kit for detecting 16 types of pathogenic microorganisms, wherein the kit comprises the primer-probe combination described in the first aspect.
[0012] In some embodiments of the present invention, the primer probe combination is prepared together with Taq enzyme and PCR reaction buffer solution into a lyophilized pellet, which contains 1.2 μL of primer probe combination, 0.5 μL of Taq enzyme and 5 μL of PCR reaction buffer solution. The Taq enzyme concentration is 1-5 U / μL, and the PCR reaction buffer solution includes 100-300 mM Tris-HCl, 200-400 mM KCl, 10-40 mM MgCl2, 50﹪DTT, and 0.2-0.6 mM dNTPs.
[0013] In some embodiments of the present invention, the method further comprises a digestion solution, a lysis solution, magnetic beads, a washing solution, a reconstitution solution, and paraffin oil; The digestion solution includes 0.1-0.5M Tris, 0.2-0.6M EDTA, and 1-4M sodium chloride; The lysis buffer includes 10-50 mM sodium citrate, 1%-5% Tween, 10-50% glycerol, and 2-6 M guanidine thiocyanate; The washing solution includes 2-5M guanidine hydrochloride, 0.1-1M Tris at pH 8.0, and 0.5-2M sodium chloride; The reconstitution solution includes 100-300 mM Tris-HCl, 200-400 mM KCl, 10-40 mM MgCl2, 50% DTT, and 0.2-0.6 mM dNTPs.
[0014] The digestion solution and lysis solution contain substances that destroy the cell wall / membrane structure of microorganisms and release internal nucleic acids; the washing solution contains a high-salt buffer system to neutralize the negative charge of humic acid and reduce PCR inhibition; the reconstitution solution contains buffer for qPCR detection, which can be used as both an eluent and a detection reagent reconstitution solution; paraffin oil is used as a liquid seal to prevent aerosol contamination.
[0015] The fourth aspect of the present invention provides a method for using the kit according to the third aspect, comprising the following steps: S1. Place the sample to be tested in a sample tube, add PBS to the sample tube, vortex mix, and transfer the turbid solution to the extraction chamber of the nucleic acid extraction and detection integrated cartridge of the nucleic acid extraction and detection integrated device; S2. Setting the extraction program on the all-in-one machine, extracting the sample in the extraction chamber to obtain the extracted product; S3. The extracted product is automatically transferred to the elution well in the integrated device, vortexed and mixed, and then diluted; S4. Transfer the diluted extract to a test tube containing a lyophilized test ball and mix thoroughly by pipetting. S5. Transfer paraffin oil to the test tube for liquid sealing; S6, performing fluorescent PCR amplification on a machine to obtain an amplification curve; S7. Interpret the amplification curve graph.
[0016] In some embodiments of the present invention, in step S2, the extraction procedure is: Digestion program for well 1: digestion solution, mixing time 600 s, liquid volume 500 µL, heating temperature 55°C; Lysis program for well 2: lysis buffer, mixing time 600 s, magnetic adsorption time 20 s, liquid volume 500 µL, heating temperature 80°C; Washing procedure for wells 3-4: wash solution, wash well 3 once, wash well 4 twice. The first wash for wells 3 and 4 was performed with a mixing time of 600 s, a magnetic adsorption time of 20 s, and a liquid volume of 500 µL. The second wash for well 4 was performed with a magnetic adsorption time of 20 s, a drying time of 240 s, and a liquid volume of 600 µL. Elution program for well 5: eluent, mixing time 300 s, magnetic adsorption time 60 s, liquid volume 100 µL, heating temperature 55 °C.
[0017] In some embodiments of the present invention, in step S6, the fluorescence PCR amplification procedure is: pre-denaturation at 95°C for 2-4 minutes; denaturation at 95°C for 5-10 seconds, annealing at 60°C for 10-30 seconds, for 40 cycles. In some embodiments of the present invention, preferably, the fluorescence PCR amplification procedure is: pre-denaturation at 95°C for 2 minutes; denaturation at 95°C for 5 seconds, annealing at 60°C for 10 seconds, for 40 cycles.
[0018] Beneficial effects of the present invention Compared with the existing technology, the present invention has the following beneficial effects: the present invention provides a set of primer-probe combinations for detecting 16 pathogenic microorganisms. The screened primer-probes have the highest sensitivity and good specificity. They are used as quadruple detection reagents, combined with optimized lysis buffer and fluorescent PCR amplification procedures, to ensure extraction effect and sensitivity. The method of the present invention combines a nucleic acid extraction and detection integrated device, and realizes full automation from nucleic acid extraction to qPCR detection. It only takes 60 minutes from extraction to qPCR detection results, shortening the detection time. BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 The figure shows the overall structure of the integrated nucleic acid extraction and detection cartridge used in the present invention; wherein, 10, reagent rack; 11, reagent tube hole; 12, reagent tube; 13, tube cover; 14, first accommodating slot; 15, pipette tip; 16, second accommodating slot; 17, single tube; 18, first heating seat; 19, second heating seat; 20, waste liquid chamber; 21, extraction chamber; 22, pressure cover; 23, buckle; 24, slot; 25, sealing rubber gasket; 26, through hole; 27, cross slot; Figure 2 The detection results of four pathogenic microorganisms, namely, colonies, total Vibrio, Helicobacter pylori, and Aeromonas hydrophila, are shown in Example 4 of the present invention; wherein FAM represents total colonies, HEX represents total Vibrio, ROX represents Helicobacter pylori, and CY5 represents Aeromonas hydrophila; Figure 3 The figure shows the detection results of four pathogenic microorganisms, Mycobacterium tuberculosis, Vibrio cholerae, Yersinia pestis, and Pseudomonas aeruginosa, in Example 4 of the present invention; wherein FAM represents Mycobacterium tuberculosis, HEX represents Vibrio cholerae, ROX represents Yersinia pestis, and CY5 represents Pseudomonas aeruginosa; Figure 4 The figure shows the detection results of four pathogenic microorganisms, Staphylococcus aureus, Mycoplasma, Chlamydia, and Rickettsia, in Example 4 of the present invention; wherein FAM represents Staphylococcus aureus, HEX represents Mycoplasma, ROX represents Chlamydia, and CY5 represents Rickettsia; Figure 5 The figure shows the detection results of four pathogenic microorganisms, Toxoplasma, Plasmodium, novel coronavirus (COVID-19), and HIV in Example 4 of the present invention; wherein FAM represents Toxoplasma, HEX represents Plasmodium, ROX represents novel coronavirus (COVID-19), and CY5 represents HIV. DETAILED DESCRIPTION
[0020] The following examples are provided to illustrate preferred embodiments of the present invention. Those skilled in the art will appreciate that the techniques disclosed in the following examples represent techniques discovered by the inventors that can be used to practice the present invention and, therefore, can be considered preferred embodiments of the present invention. However, those skilled in the art will appreciate from this disclosure that many modifications may be made to the specific embodiments disclosed herein while still achieving the same or similar results without departing from the spirit or scope of the present invention.
[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one skilled in the art to which this invention belongs, and the disclosures herein and the materials they cite are hereby incorporated by reference. Those skilled in the art will recognize or be able to ascertain, through routine experimentation, many technical equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the claims.
[0022] The technical solution of the present application will be further described in detail below in conjunction with specific implementation methods.
[0023] Example 1 Screening of primers and probes for 16 pathogenic microorganisms The pathogenic microorganisms that need to be tested include total colony counts, total Vibrio, Helicobacter pylori, Aeromonas hydrophila, Mycobacterium tuberculosis (causing tuberculosis), Vibrio cholerae, Yersinia pestis, Pseudomonas aeruginosa, Staphylococcus aureus (causing multiple infections), Mycoplasma, Chlamydia, Rickettsia, Toxoplasma, Plasmodium, new coronavirus (COVID-19), HIV, etc. 16 types.
[0024] Three sets of primers and probes were designed for the above 16 pathogenic microorganisms. The information of the three sets of primers and probes are shown in Tables 1 to 3 below.
[0025] Table 1 Information of the first set of primers and probes for 16 pathogenic microorganisms
[0026] Table 2 Information of the second set of primers and probes for 16 pathogenic microorganisms
[0027] Table 3 Information of the third set of primers and probes for 16 pathogenic microorganisms
[0028] The primers and probes with the same sequence code number for each microorganism species in the above table were combined into a set of combinations for single-plex detection. The test results are shown in Table 4.
[0029] Table 4 Test results of different primer combinations
[0030] The results showed that the first set of primers and probes had the best sensitivity.
[0031] Subsequently, quadruple detection of 16 pathogenic microorganisms was performed using the first set of primer-probe combinations.
[0032] Example 2 Optimization of lysate The following two lysis solutions were used to extract 16 pathogenic microorganisms; Lysis buffer 1: contains 40 mM sodium citrate, 2% Tween, 10% glycerol, and 4 M guanidine thiocyanate; The specific preparation method is as follows: accurately weigh 11.76g sodium citrate, 20mL Tween, 100mL glycerol and 472.64g guanidine thiocyanate in a 1L beaker, add water to dissolve and then adjust the volume to 1L; Lysis buffer 2: contains 40 mM sodium citrate, 2% Tween, and 6 M guanidine thiocyanate; The specific preparation method is as follows: accurately weigh 11.76g sodium citrate, 20mL Tween and 708.96g guanidine thiocyanate in a 1L beaker, add water to dissolve and then adjust the volume to 1L; The results of qPCR amplification of the extracted products are shown in Table 5.
[0033] Table 5 Extraction effects of different lysis buffers on 16 pathogenic microorganisms
[0034] The results showed that lysis buffer 1 could successfully lyse and extract nucleic acids of bacteria, parasites and viruses; while lysis buffer 2 could not lyse parasites and viruses, resulting in the inability to extract nucleic acids of parasites and viruses.
[0035] Example 3 Optimization of qPCR detection procedure Optimize the gradient of pre-denaturation time, deformation time, and annealing time in the qPCR detection program: qPCR detection program: 95°C 4 min / 3 min / 2 min; 95°C 10 s / 8 s / 5 s, 60°C 30 s / 20 s / 10 s, 40 cycles.
[0036] The test results are shown in Table 6.
[0037] Table 6 Detection results of different qPCR detection procedures
[0038] The results showed that, based on the gradient optimization of pre-denaturation, denaturation, and annealing times, the Ct values for pre-denaturation times of 4 minutes, 3 minutes, and 2 minutes were similar; the Ct values for denaturation times of 10 seconds, 8 seconds, and 5 seconds were similar; and the Ct values for annealing times of 30 seconds, 20 seconds, and 10 seconds were similar. In summary, to shorten this experiment, a 2-minute pre-denaturation, 5-second denaturation, and 10-second annealing schedule was the preferred final detection schedule.
[0039] Example 416 Quadruple Detection Method for Pathogenic Microorganisms Use an integrated nucleic acid extraction and detection cartridge, extraction reagents, and four tubes of 4-plex fluorescent PCR detection reagents for detecting different pathogens to perform corresponding detection operations.
[0040] Nucleic acid extraction and detection integrated cartridges such as Figure 1 Schematic diagram, including: reagent rack 10; reagent tube hole 11; reagent tube 12; tube cover 13; first receiving slot 14; pipette tip 15; second receiving slot 16; single strip tube 17; first heating block 18; second heating block 19; waste liquid chamber 20; extraction chamber 21; pressure cover 22; buckle 23; slot 24; sealing gasket 25; through hole 26; cross groove 27. Specifically, extraction reagents are added to the single strip tubes of the integrated nucleic acid extraction and detection cartridge; four reagent tubes are added to each of the four 4-plex fluorescent PCR detection reagent tubes, and the final extraction product is produced in the extraction chamber.
[0041] The single tube of the extraction reagent contains digestion buffer, lysis buffer, magnetic beads, wash buffer 1, wash buffer 2, resolvent, and paraffin oil. The digestion buffer and lysis buffer contain enzymes that disrupt microbial cell walls and membranes, releasing nucleic acids within. The wash buffer contains a high-salt buffer system to neutralize the negative charge of humic acid and reduce PCR inhibition. The resolvent contains the buffer for qPCR detection, which serves as both an eluent and a resolvent for the detection reagents. Paraffin oil serves as a liquid seal to prevent aerosol contamination.
[0042] Digestion solution: 0.2 M Tris, 0.5 M EDTA, 3 M NaCl; Lysis buffer: 40 mM sodium citrate, 2% Tween, 10% glycerol, 4 M guanidine thiocyanate; Wash buffer 1: 2 M guanidine hydrochloride, 0.1 M Tris (pH 8.0), 1 M sodium chloride; Wash Buffer 2: 2 M guanidine hydrochloride, 0.1 M Tris (pH 8.0), 1 M sodium chloride; The resolvent is PCR reaction buffer, which contains: 300mM Tris-HCl, 300mM KCl, 30mM MgCl2, 50﹪DTT, and 0.6mM dNTPs.
[0043] The procedure for extracting pathogenic microorganisms is shown in Table 7.
[0044] Table 7 Procedure for extracting pathogenic microorganisms
[0045] By adopting the above technical solution, each tube of the detection reagent tube contains a freeze-dried ball, which contains 1.2 μL of primer-probe combination, 0.5 μL of Taq enzyme and 5 μL of PCR reaction buffer solution.
[0046] The concentration of Taq enzyme is 1 U / μL, 2 U / μL, 3 U / μL, 4 U / μL, and 5 U / μL, with 5 U / μL being the optimal formulation.
[0047] The buffer required for the PCR reaction contains 100-300mM Tris-HCl, 200-400mM KCl, 10-40mM MgCl2, 50% DTT, and 0.2-0.6mM dNTPs. The optimal formulation is 300mM Tris-HCl, 300mM KCl, 30mM MgCl2, and 0.6mM dNTPs.
[0048] The fluorescent channels selected by the probe are FAM, HEX, CY5, and ROX. The channel selected for colonies, Mycobacterium tuberculosis, Staphylococcus aureus, and Toxoplasma is the FAM channel; the channel selected for total Vibrio, Vibrio cholerae, Mycoplasma, and Plasmodium is HEX; the channel selected for Helicobacter pylori, Yersinia pestis, Chlamydia, and the new coronavirus (COVID-19) is CY5; the channel selected for Aeromonas hydrophila, Pseudomonas aeruginosa, Rickettsia, and HIV is ROX.
[0049] There are four test tubes in total, including one for the quadruple fluorescent PCR test for total bacterial counts, total Vibrio, Helicobacter pylori, and Aeromonas hydrophila; one for the quadruple fluorescent PCR test for Mycobacterium tuberculosis, Vibrio cholerae, Yersinia pestis, and Pseudomonas aeruginosa; one for the quadruple fluorescent PCR test for Staphylococcus aureus, Mycoplasma, Chlamydia, and Rickettsia; and one for the quadruple fluorescent PCR test for Toxoplasma, Plasmodium, novel coronavirus (COVID-19), and HIV. These four test tubes detect 16 pathogens.
[0050] By adopting the above technical scheme, the qPCR detection program is 95°C for 2 min; 95°C for 10 s, 60°C for 20 s, and 40 cycles.
[0051] In this embodiment, bacteria, Mycobacterium tuberculosis, Staphylococcus aureus, Toxoplasma gondii, Vibrio spp., Vibrio cholerae, Mycoplasma, Plasmodium, Helicobacter pylori, Yersinia pestis, Chlamydia, novel coronavirus (COVID-19), Aeromonas hydrophila, Pseudomonas aeruginosa, Rickettsia, HIV and other pseudoviruses are added to the water body to form the contaminated water body as the sample to be tested.
[0052] The specific detection process is as follows: (1) Place the 0.45µm filter membrane in the filter head interlayer, tighten the filter head assembly, and filter the water to be tested using a syringe or a suction pump to collect the sample microorganisms on the filter membrane. Use sterile tweezers to remove the filter membrane, cut it into pieces, and transfer it to a 2mL centrifuge tube; (2) Add 500 μL of PBS to the centrifuge tube containing the filter membrane, vortex to mix, and add 200 μL of the turbid solution to the extraction chamber of the integrated nucleic acid extraction and detection cartridge; (3) Set the extraction procedure according to Table 2. After the sample is extracted in the extraction chamber, 100 μL of product is obtained; (4) The instrument automatically transfers 20 μL of the extracted product to the elution well, vortexes to mix, and then dilutes the extracted product; (5) The instrument automatically transfers 25 μL into four quadruple assay reagent tubes and mixes thoroughly by pipetting; (6) Transfer 25 μL of paraffin oil to four quadruple assay reagent tubes and seal them; (7) Conduct fluorescence PCR detection on the machine, (8) Interpret the amplification curve, such as Figures 2 to 5 shown.
[0053] The results showed that the method of this application can simultaneously detect 16 pathogenic microorganisms in water samples. The total time from extraction to detection is no more than 60 minutes.
[0054] Comparative Example Commercial kits and the method of the present invention were used to simultaneously detect 2 samples of each of 16 pathogens. The extraction reagent used was the Ark Biomagnetic Bead Aquatic Animal Pathogen Nucleic Acid Extraction Kit, and the detection reagents used were Novozymes AceQ® UniversalU + Probe Master Mix V2, Taq Pro Universal SYBR qPCR Master Mix, and HIV detection kit.
[0055] (1) Extract the nucleic acid of the sample to be tested according to the instructions of the Ark Biomagnetic Bead Aquatic Animal Pathogen Nucleic Acid Extraction Kit; (2) qPCR detection of 16 pathogens was performed according to the instructions of Novozymes AceQ® Universal U + Probe Master Mix V2, Taq Pro Universal SYBR qPCR Master Mix, and HIV detection kit. The primers and probes used in the control group were those in the published data or the kit. The primer and probe information is shown in Table 8.
[0056] Table 8 Primer and probe information in the control group
[0057] HIV primer and probe information is not disclosed in the kit.
[0058] (3) The detection results of the commercial kit and the method of the present invention are shown in Table 9.
[0059] Table 9 Comparison of detection results between commercial kit and the method of the present invention
[0060] Results showed that the method of this invention achieved a Ct value 1-2 times lower than that of conventional commercial kits, demonstrating its higher sensitivity. Furthermore, the method of this invention is fully automated, from nucleic acid extraction to qPCR detection, taking only 60 minutes from extraction to qPCR results. Conventional kit methods require over 2 hours for the entire process.
[0061] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the present application.
Claims
1. A primer-probe combination for detecting 16 pathogenic microorganisms, characterized in that: The primer-probe combination comprises: The forward primer for targeted amplification of total colonies is shown in SEQ ID NO.1, the reverse primer is shown in SEQ ID NO.2, and the probe for detecting total colonies is shown in SEQ ID NO.3; The forward primer for targeted amplification of total Vibrio is shown in SEQ ID NO.4, the reverse primer is shown in SEQ ID NO.5, and the probe for detecting total Vibrio is shown in SEQ ID NO.6; The forward primer for targeted amplification of Helicobacter pylori is shown in SEQ ID NO.7, the reverse primer is shown in SEQ ID NO.8, and the probe for detecting Helicobacter pylori is shown in SEQ ID NO.9; The forward primer for targeted amplification of Aeromonas hydrophila is shown in SEQ ID NO.10, the reverse primer is shown in SEQ ID NO.11, and the probe for detecting Helicobacter pylori is shown in SEQ ID NO.12; The forward primer for targeted amplification of Mycobacterium tuberculosis is shown in SEQ ID NO. 13, the reverse primer is shown in SEQ ID NO. 14, and the probe for detecting Mycobacterium tuberculosis is shown in SEQ ID NO. 15; The forward primer for targeted amplification of Vibrio cholerae is shown in SEQ ID NO.16, the reverse primer is shown in SEQ ID NO.17, and the probe for detecting Vibrio cholerae is shown in SEQ ID NO.18; The forward primer for targeted amplification of Yersinia pestis is shown in SEQ ID NO. 19, the reverse primer is shown in SEQ ID NO. 20, and the probe for detecting Yersinia pestis is shown in SEQ ID NO. 21; The forward primer for targeted amplification of Pseudomonas aeruginosa is shown in SEQ ID NO. 22, the reverse primer is shown in SEQ ID NO. 23, and the probe for detecting Pseudomonas aeruginosa is shown in SEQ ID NO. 24; The forward primer for targeted amplification of Staphylococcus aureus is shown in SEQ ID NO. 25, the reverse primer is shown in SEQ ID NO. 26, and the probe for detecting Staphylococcus aureus is shown in SEQ ID NO. 27; The forward primer for targeted amplification of Toxoplasma gondii is shown in SEQ ID NO. 28, the reverse primer is shown in SEQ ID NO. 29, and the probe for detecting Toxoplasma gondii is shown in SEQ ID NO. 30; The forward primer for targeted amplification of Plasmodium is shown in SEQ ID NO.31, the reverse primer is shown in SEQ ID NO.32, and the probe for detecting Plasmodium is shown in SEQ ID NO.33; The forward primer for targeting mycoplasma is shown in SEQ ID NO. 34, the reverse primer is shown in SEQ ID NO. 35, and the probe for detecting Plasmodium is shown in SEQ ID NO. 36; The forward primer for targeted amplification of Chlamydia is shown in SEQ ID NO. 37, the reverse primer is shown in SEQ ID NO. 38, and the probe for detecting Chlamydia is shown in SEQ ID NO. 39; The forward primer for targeted amplification of Rickettsia is shown in SEQ ID NO.40, the reverse primer is shown in SEQ ID NO.41, and the probe for detecting Rickettsia is shown in SEQ ID NO.42; The forward primer for targeted amplification of the novel coronavirus is shown in SEQ ID NO. 43, the reverse primer is shown in SEQ ID NO. 44, and the probe for detecting the novel coronavirus is shown in SEQ ID NO. 45; The forward primer for targeted amplification of HIV is shown in SEQ ID NO.46, the reverse primer is shown in SEQ ID NO.47, and the probe for detecting HIV is shown in SEQ ID NO.
48.
2. A primer-probe combination for detecting 16 pathogenic microorganisms according to claim 1, characterized in that: The primer-probe combination is divided into four groups: total colonies, total Vibrio, Helicobacter pylori, and Aeromonas hydrophila are one group; Mycobacterium tuberculosis, Vibrio cholerae, Yersinia pestis, and Pseudomonas aeruginosa are one group; Staphylococcus aureus, Mycoplasma, Chlamydia, and Rickettsia are one group; Toxoplasma, Plasmodium, new coronavirus, and HIV are one group.
3. The primer-probe combination for detecting 16 pathogenic microorganisms according to claim 1, characterized in that: In the primer-probe combination, the 5' end of the probe is connected to a fluorescent group, and the 3' end is connected to a quenching group. The fluorescent group is selected from one of FAM, HEX, CY5, and ROX. The fluorescent labels used on the probes of the same group of pathogenic microorganisms are different.
4. Use of the primer-probe combination according to any one of claims 1 to 3 in the preparation of products for detecting 16 types of pathogenic microorganisms.
5. A kit for detecting 16 pathogenic microorganisms, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 3.
6. A kit for detecting 16 pathogenic microorganisms according to claim 5, characterized in that: The primer-probe combination, enzyme, and PCR reaction buffer solution are prepared into a lyophilized pellet, which contains 1.2 μL of the primer-probe combination, 0.5 μL of Taq enzyme, and 5 μL of PCR reaction buffer solution. The Taq enzyme concentration is 1-5 U / μL, and the PCR reaction buffer solution includes 100-300 mM Tris-HCl, 200-400 mM KCl, 10-40 mM MgCl2, 50﹪DTT, and 0.2-0.6 mM dNTPs.
7. A kit for detecting 16 pathogenic microorganisms according to claim 6, characterized in that: It also contains digestion solution, lysis solution, magnetic beads, washing solution, reconstitution solution and paraffin oil; The digestion solution includes 0.1-0.5M Tris, 0.2-0.6M EDTA, and 1-4M sodium chloride; The lysis buffer includes 10-50 mM sodium citrate, 1%-5% Tween, 10-50% glycerol, and 2-6 M guanidine thiocyanate; The washing solution includes 2-5 M guanidine hydrochloride, 0.1-1 M Tris at pH 8.0, and 0.5-2 M sodium chloride; The reconstitution solution includes 100-300 mM Tris-HCl, 200-400 mM KCl, 10-40 mM MgCl2, 50% DTT, and 0.2-0.6 mM dNTPs.
8. A method for using the kit according to any one of claims 4 to 6, characterized in that: The steps include: S1. Place the sample to be tested in a sample tube, add PBS to the sample tube, vortex mix, and transfer the turbid solution to the extraction chamber of the nucleic acid extraction and detection integrated cartridge of the nucleic acid extraction and detection integrated device; S2. Setting the extraction program on the all-in-one machine, extracting the sample in the extraction chamber to obtain the extracted product; S3. The extracted product is automatically transferred to the elution well in the integrated device, vortexed and mixed, and then diluted; S4. Transfer the diluted extract to a test tube containing a lyophilized test ball and mix thoroughly by pipetting. S5. Transfer paraffin oil to the test tube for liquid sealing; S6, performing fluorescent PCR amplification on a machine to obtain an amplification curve; S7. Interpret the amplification curve graph.
9. The method for using the kit according to claim 5, wherein: In step S2, the extraction procedure is: Digestion program for well 1: digestion solution, mixing time 600 s, liquid volume 500 µL, heating temperature 55°C; Lysis program for well 2: lysis buffer, mixing time 600 s, magnetic adsorption time 20 s, liquid volume 500 µL, heating temperature 80°C; Washing procedure for wells 3-4: wash solution, wash well 3 once, wash well 4 twice. The first wash for wells 3 and 4 was performed with a mixing time of 600 s, a magnetic adsorption time of 20 s, and a liquid volume of 500 µL. The second wash for well 4 was performed with a magnetic adsorption time of 20 s, a drying time of 240 s, and a liquid volume of 600 µL. Elution program for well 5: eluent, mixing time 300 s, magnetic adsorption time 60 s, liquid volume 100 µL, heating temperature 55 °C.
10. The method for using the kit according to claim 5, characterized in that: In step S6, the fluorescence PCR amplification program is: pre-denaturation at 95°C for 2-4 min; denaturation at 95°C for 5-10 s, annealing at 60°C for 10-30 s, for 40 cycles.