Primer probe composition and method for simultaneously detecting five pneumonia pathogens on site

Abstract

The invention discloses a primer probe composition and a method for simultaneously detecting five pneumonia pathogens on site, and relates to the technical field of biological detection. The invention provides a primer probe combination for on-site detection of pneumonia pathogens. The primer probe combination comprises a primer pair and a probe for detecting streptococcus pneumoniae, the primer pair and the probe are used for detecting klebsiella pneumoniae; the primer pair and the probe are used for detecting legionella pneumophila; the primer pair and the probe are used for detecting chlamydia pneumoniae; the invention discloses a primer pair and a probe for detecting mycoplasma pneumoniae. The method for detecting the pneumonia pathogens by adopting the primer probe combination and the kit disclosed by the invention integrates sample pretreatment, nucleic acid extraction, purification, amplification and detection, has better sensitivity and accuracy, is simple, convenient, rapid and time-saving to operate, and plays an important role in on-site rapid detection of the pneumonia pathogens.

Description

Technical Field

[0001] The present invention relates to the field of biological detection technology, and in particular to a primer-probe combination and a kit for on-site detection of pneumonia pathogens. Background Art

[0002] Respiratory infections are of paramount public health importance. Their impact not only affects individual health but also poses multiple threats to society as a whole. They cause millions of deaths worldwide each year. Furthermore, infections can lead to serious complications such as pneumonia and myocarditis, resulting in sustained impacts on the workforce, medical resources, and socioeconomic stability. Therefore, scientifically preventing and controlling respiratory infections is crucial for maintaining public health and social development.

[0003] Streptococcus pneumoniae ( Streptococcus pneumoniae , hereinafter referred to as SP) is a Gram-positive coccus with a genome size of approximately 2-2.5Mbp, encoding approximately 2,000 genes. The diameter of Streptococcus pneumoniae is approximately 1μm, and the genome is often arranged in pairs. Streptococcus pneumoniae mainly causes lobar pneumonia in humans, and some healthy people carry virulent Streptococcus pneumoniae in their upper respiratory tract. Klebsiella pneumoniae ( Klebsiellapneumoniae , hereinafter referred to as KP) is a Gram-negative bacillus. The typical genome size of K. pneumoniae is about 5-6Mbp, encoding about 5000-6000 genes. The typical characteristics of pneumonia caused by K. pneumoniae are necrotic damage to the alveolar space, often forming cavities, and accompanied by coughing up blood-streaked sputum. Legionella pneumophila Chlamydia pneumoniae (Lpn) is a flagellated, Gram-negative, polymorphic, short cocci of the genus Legionella. Its genome size is approximately 3.4Mb and it was first discovered in 1977. It is one of the main pathogens causing Legionnaires' disease, which can cause severe pneumonia. Chlamydiapneumonia , hereinafter referred to as CP) is a human respiratory pathogen found in all age groups worldwide. It is an obligate intracellular bacterium that can only be isolated in tissue culture, but culture is not widely available. Chlamydia pneumoniae infection is a common cause of community-acquired pneumonia in children and adults. Mycoplasma Pneumonia , hereinafter referred to as MP ） It is the causative agent of human Mycoplasma pneumoniae, a common cause of community-acquired pneumonia and lower respiratory tract infections.

[0004] Methods for detecting pneumonia pathogens primarily include serological testing, antigen testing, and nucleic acid testing. Nucleic acid testing is widely used due to its rapidity and accuracy. Nucleic acid-based detection methods include quantitative real-time PCR (qPCR), digital PCR (dPCR), isothermal amplification, and next-generation sequencing (NGS). qPCR is the most commonly used technique, enabling the detection, identification, and quantification of pathogens using SYBR Green or TaqMan probes. For example, patent CN118853914A discloses a method for the simultaneous detection of six respiratory pathogens. There are many similar patents, but most require nucleic acid extraction followed by quantitative PCR amplification, a complex and time-consuming process.

[0005] Based on the above problems, the inventors have conducted several years of research and development and disclosed a new type of on-site rapid nucleic acid detection equipment in the prior application CN113174324A. The equipment is a "handheld real-time fluorescence PCR all-in-one machine P1000F". After temperature control optimization and upgrading, it can complete rapid nucleic acid detection within 35 minutes, and can realize on-site automated detection of "sample in, result out".

[0006] To address the challenges of sample pretreatment, the inventors have developed a simple and fast-operating sample processing tube (CN220136779U). Building on the sample processing and detection equipment, the present inventors have further developed an invention that better complements this equipment: a primer-probe combination and an integrated closed microfluidic chip kit for the combined detection of five pneumonia pathogens. This kit extracts, purifies, amplifies, and detects nucleic acid from samples on an integrated chip, enabling rapid, on-site point-of-care (POCT) testing. Summary of the Invention

[0007] The purpose of the present invention is to provide a primer-probe combination and a kit for the on-site simultaneous detection of five pneumonia pathogens. The primer-probe combination and the kit described in the present invention are used to detect pneumonia pathogens with good sensitivity and accuracy. The method is simple to operate, fast and time-saving.

[0008] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows: In one aspect, the present invention provides a primer-probe combination for on-site detection of pneumonia pathogens, the primer-probe combination comprising: primer pairs and probes for detecting Streptococcus pneumoniae, whose sequences are respectively shown in SEQ ID NOs: 1-3; primer pairs and probes for detecting Klebsiella pneumoniae, whose sequences are respectively shown in SEQ ID NOs: 4-6; primer pairs and probes for detecting Legionella pneumophila, whose sequences are respectively shown in SEQ ID NOs: 7-9; primer pairs and probes for detecting Chlamydia pneumoniae, whose sequences are respectively shown in SEQ ID NOs: 10-12; and primer pairs and probes for detecting Mycoplasma pneumoniae, whose sequences are respectively shown in SEQ ID NOs: 13-15.

[0009] Specifically, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a quencher group.

[0010] More specifically, the fluorescent reporter group is selected from one or more of FAM, ROX, HEX, CY5, VIC, TET, JOE, CY3, CY7, RED610, TexasRed, RED670, NED, AMCA, PacificBlue, ATTO425, BODIPYFL, AlexaFluor488, YakimaYellow, Quasar570, AquaPhluor593, ATTO590, and CY5.5.

[0011] More specifically, the fluorescent reporter group is selected from one or more of ATTO425, FAM, HEX, CY3, CY5, CY5.5, VIC, JOE, and ROX.

[0012] More specifically, the quencher group is a fluorescence quencher group.

[0013] More specifically, the fluorescence quenching group is selected from one or more of 6-TAMRA, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, QYS-7, and SQ1.

[0014] More specifically, the fluorescence quenching group is selected from one or more of BHQ2, BHQ3, SQ1, and MGB.

[0015] In another aspect, the present invention provides the use of the above primer-probe combination in the preparation of a product for detecting Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae and Mycoplasma pneumoniae.

[0016] Specifically, the products include independent detection reagents, microfluidic chips and test kits.

[0017] In another aspect, the present invention provides a kit for on-site detection of pneumonia pathogens, the kit comprising the above-mentioned primer-probe combination.

[0018] Specifically, the kit is an integrated closed microfluidic chip kit.

[0019] More specifically, the integrated closed microfluidic chip kit includes purified reagents and air-dried qPCR reagents. Specifically, the purified reagents and air-dried qPCR reagents can be transported and stored at room temperature.

[0020] More specifically, the purification reagent includes a lysis solution, a washing solution, an eluent and magnetic beads, the lysis solution and the washing solution include guanidine hydrochloride, sodium acetate, TritonX-100 and 1,3-butanediol, the eluent includes Tris-HCl, and the magnetic beads are dried magnetic beads.

[0021] More specifically, the lysis solution and washing solution include 4.2M guanidine hydrochloride, 0.28M sodium acetate (pH 4.7), 1.4% Triton X-100 and 30% 1,3-butanediol, and the eluent includes 10mM Tris-HCl (pH 8.5).

[0022] Specifically, the kit completes the nucleic acid extraction, purification, amplification and detection of the sample on an integrated chip.

[0023] Specifically, the microfluidic chip kit can be used for the P1000F rapid nucleic acid detection device.

[0024] In another aspect, the present invention provides a method for on-site detection of pneumonia pathogens, the method comprising preparation of an integrated closed microfluidic chip kit, sample pretreatment, and rapid device detection; Specifically, the method comprises the following steps: (1) The above primer-probe combination is prepared into an integrated closed microfluidic kit; (2) Add samples containing Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae into a sample processing tube for processing to obtain a liquid to be tested; (3) Drop the liquid to be tested into the integrated closed microfluidic reagent kit; (4) Place the test kit with the liquid to be tested on the testing equipment for testing and analysis.

[0025] Specifically, the step (2) is as follows: adding a sample pretreatment solution and abrasive particles into a sample processing tube; the sample pretreatment solution includes NaOH, NaCl and TritonX-100; More specifically, the concentration range of NaOH is 1%-5%, the concentration range of NaCl is 30mM-1.4M, the concentration range of TritonX-100 is 0.1%-2%, and the diameter range of the grinding particles is 0.1 micron-1 mm.

[0026] More specifically, the concentration of NaOH in the sample pretreatment solution ranges from 3% to 5%, the concentration of NaCl ranges from 350 mM to 700 mM, and the concentration of Triton X-100 ranges from 0.4% to 0.8%.

[0027] Specifically, the sample described in step (2) is a sample of sputum, alveolar lavage fluid, oropharyngeal / nasopharyngeal swab preservation fluid, tongue swab preservation fluid, drainage fluid, etc. containing Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, or Mycoplasma pneumoniae.

[0028] Specifically, the step (4) includes: 1) Turn on the rapid detection device; 2) Scan the QR code; 3) Insert the integrated microfluidic chip kit; 4) Run the test; 5) When finished, check the test results and amplification curve.

[0029] According to some embodiments of the present invention, the method comprises the following steps: (1) The above primer-probe combination is used to prepare an integrated closed microfluidic kit; (2) Rapid sample pre-processing using a sample processing tube; (3) Using the purification reagents on the chip to lyse the sample and extract and purify the nucleic acid; (4) using the above primer-probe combination to rapidly amplify the sample nucleic acid extracted and purified in step (3); (5) Analyze the amplification results.

[0030] In another aspect, the present invention provides use of the above primer-probe combination, the above kit, or the above method in detecting Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

[0031] The beneficial effects of the present invention are: The present invention provides a primer-probe combination, an integrated microfluidic chip kit, and a method for detecting pneumonia pathogens. The method for detecting pneumonia pathogens using the primer-probe combination and the kit of the present invention integrates sample pretreatment, nucleic acid extraction, purification, amplification, and detection, has good sensitivity and accuracy, and the method is simple to operate, fast, and time-saving. At the same time, when the sample processing tube provided by the present invention is used to process sputum samples, the sputum sample can be liquefied in 1 minute, greatly shortening the time for liquefying the sputum sample. In addition, the method provided by the present invention can simultaneously detect Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae, and plays an important role in the on-site rapid detection of pneumonia pathogens. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is the test result chart of the mixed quality control product of Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

[0033] Figure 2 This is a graph showing the test results of mixed quality control products and liquefied sputum samples of Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

[0034] Figure 3 This is a graph showing the test results of the mixed quality control products of Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae in Comparative Example 1.

[0035] Figure 4 This is a physical picture of the integrated microfluidic chip and sample processing tube. DETAILED DESCRIPTION

[0036] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0037] The microfluidic chip device for nucleic acid detection of the present invention is detailed in CN113278509A, the magnetic bead drying method is detailed in patent CN113005117A, the sample processing tube is detailed in patent CN220136779U, and the overall device, i.e., the PCR reaction device, is detailed in patent CN113174324A. The actual picture of the integrated microfluidic chip and sample processing tube is as follows Figure 4 shown.

[0038] Example 1 Primer-probe combination for rapid on-site detection of Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae Primer and probe sequences were designed based on the specific sequences of Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

[0039] Among them, the primer probe sequence of Streptococcus pneumoniae is: SP-F: 5'-GCCTCTATCCTGGAGCACTTCTC-3' (SEQ ID NO: 1); SP-R: 5'-CGCAAGAAGAGTGGGATTATTCTC-3' (SEQ ID NO: 2); SP-P: 5'-AGTGGATGAGACCTTG-3' (SEQ ID NO: 3).

[0040] The primer and probe sequences for Klebsiella pneumoniae are: KP-F: 5'-GCCATCTTTCATCAACCATTTCT-3' (SEQ ID NO: 4); KP-R: 5'-AGAAAATTTGGCTGTAAAGGGTACA-3' (SEQ ID NO: 5); KP-P: 5'-AGACTGTAAAGATACATAAACC-3' (SEQ ID NO: 6).

[0041] The primer and probe sequences for Legionella pneumophila are: Lpn-F: 5'-CCCGGTAAATCGGATACAGTCA-3' (SEQ ID NO:7); Lpn-R: 5'-GTCAAAAACGGTACCATCAATCAG-3' (SEQ ID NO: 8); Lpn-P: 5'-TGTCGAGTACACTGGTC-3' (SEQ ID NO: 9).

[0042] The primer and probe sequences for Chlamydia pneumoniae are: CP-F: 5'-CGCATTGCTCAGCCAAAAC-3' (SEQ ID NO: 10); CP-R: 5'-AGTAAAGAAGGGTTCCATGCAGTT-3' (SEQ ID NO: 11); CP-P: 5'-ACCTACAGCTGTTTTAAA-3' (SEQ ID NO: 12).

[0043] The primer and probe sequences for Mycoplasma pneumoniae are: MP-F: 5'-AAGCACGAGTGACGGAAACAC-3' (SEQ ID NO: 13); MP-R: 5'-CACCACATCATTCCCCGTATT-3' (SEQ ID NO: 14); MP-P: 5'-TCCTCCACCAACAACCTCGCGC-3' (SEQ ID NO: 15).

[0044] The primer-probe combination for detecting pneumonia pathogens of the present invention is used for detecting Streptococcus pneumoniae, Streptococcus pneumoniae, and Streptococcus pneumoniae in combination or alone. Rabbiella, Legionella pneumophila, Chlamydia pneumoniae, Mycoplasma pneumoniae.

[0045] In the embodiment, the Streptococcus pneumoniae probe is labeled with ATTO 425, and the quenching group is MGB, the Klebsiella pneumoniae probe is labeled with FAM, and the quenching group is MGB, the Legionella pneumophila probe is labeled with HEX, and the quenching group is MGB, the Chlamydia pneumoniae probe is labeled with CY5, and the quenching group is MGB, and the Mycoplasma pneumoniae probe is labeled with CY5.5, and the quenching group is BHQ3.

[0046] Example 2 A microfluidic chip kit for detecting pneumonia pathogens (1) The kit includes the primer-probe combination described in Example 1, with final concentrations as shown in Table 1 below.

[0047] Table 1 Primer and probe concentrations

[0048] (2) The kit also includes purification reagents and air-drying reagents.

[0049] 1) The purification reagents include 450 μL of lysis buffer, 450 μL of wash buffer, 850 μL of eluent, and 15 μL of magnetic beads. The lysis buffer and wash buffer include 4.2 M guanidine hydrochloride, 0.28 M sodium acetate (pH 4.7), 1.4% Triton X-100, and 30% 1,3-butanediol. The eluent includes 10 mM Tris-HCl (pH 8.5). The purification reagents are added to the sample loading layer of the chip. The magnetic bead drying method is described in patent CN113005117A.

[0050] 2) The 50 μL air-dried reagent system is shown in Table 2 below.

[0051] Table 2 Air-dried reagent system

[0052] Add the reagents in Table 2 above to the reaction chamber of the integrated microfluidic chip, place the chip in an air drying oven, and air-dry according to the Air-Dryable qPCR Mix instructions.

[0053] Example 3 Preparation of sample processing tube (1) Install the sample processing tube; (2) Add about 1 g of corundum to a tube and add 2 mL of sample pretreatment solution (4% NaOH, 500 mM NaCl, 0.5% Triton X-100) to the tube; (3) Place the tube in the tooling position of the precision manual press and wrap it with aluminum foil at 160°C.

[0054] Example 4 Sputum Sample Detection Method (1) Tear off the aluminum foil protective film of the sample processing tube, place it vertically, add no less than 1 mL of sputum sample into the sample processing tube, and tighten the tube cap; (2) Keep the tube upright and rub it 5-10 times to ensure that the sample is fully in contact with the grinding material and that the grinding is sufficient; (3) Open the outer packaging, take out the reagent chamber and reaction layer of the chip, tear off the sealing strip on the back of the reagent chamber, hold the reagent chamber and reaction layer so that the head of the reaction layer is vertically upward, press hard to puncture the reagent chamber until the upper and lower layers of the chip are seamlessly connected, and complete the assembly; (4) Unscrew the sample chamber cover, add 2-3 drops (50 μL) of sample grinding solution into the sample chamber, and tighten the sample chamber cover; (5) Press and hold the device power button for more than 3 seconds to turn on the device, and click the "Detect" button on the homepage; (6) After waiting for 3 seconds, you will see the red light flashing in the scanning window and scan the chip QR code; (7) Scan the sample information or skip according to the on-screen prompts; (8) Follow the on-screen instructions to open the hatch, insert the chip, and close the hatch; (9) Click the "Run" button to start the test; (10) After the run is completed, check the test results.

[0055] The entire process of testing sputum samples for pneumonia pathogens, from sample processing, nucleic acid extraction and purification to amplification of test results, takes about 36 minutes.

[0056] Experimental Example 1 Sensitivity Detection Positive quality control samples were used in all experimental tests. Specific information is shown in Table 3. Five nucleic acid quality control samples were diluted to 1×104 copies / mL, 1×103 copies / mL, and 1×102 copies / mL, respectively. Nucleic acid testing was performed on these samples according to the systems and procedures described in Examples 1-4. The test results are shown in Table 4 below.

[0057] Table 3

[0058] Table 4 Sensitivity test results

[0059] As can be seen from Table 4 above, the primer composition and kit described in the present application have good sensitivity and can detect 100 copies / mL of nucleic acid quality control products.

[0060] Experimental Example 2 Specificity Detection The specific detection quality control products in Table 5 were respectively taken, and the nucleic acid detection of the above samples was performed according to the system and steps of the above Examples 1-4. The specific detection results are shown in Table 6 below, and the test results were all negative.

[0061] Table 5

[0062] Table 6 Specificity test results

[0063] As shown in Table 6, the primer-probe combination and the kit thereof of the present invention have good specificity.

[0064] Experimental Example 3 Repeatability Test Nucleic acid quality control samples for Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae were diluted to 1×102 copies / mL. Nucleic acid testing was performed on the extracted samples according to the system and steps of Examples 1-4 above, repeated 10 times. The amplification Ct values ​​and CV values ​​are shown in Table 7 below.

[0065] Table 7 Repeatability test results

[0066] As shown in Table 7, the pneumonia pathogen primer-probe composition of the present invention can accurately detect Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae, and can be used for rapid detection of pneumonia pathogens on site.

[0067] Experimental Example 4: Simulation Sample Testing In order to investigate the effect of sample type on the test results, a mixed positive control of Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae was diluted to 1×103 copies / mL and divided into two parts. One part was added with sputum liquefied by the sample treatment tube of the present invention, and the other part was not treated as a reference. The above samples were tested according to the system and steps of Examples 1-4 above. The amplification Ct values ​​are shown in Table 8 below. The amplification results are shown in Table 8 below. Figure 1 and Figure 2 .

[0068] Table 8 Simulation sample test results

[0069] As shown in Table 8, the primer-probe combinations for the five pneumonia pathogens described in the present invention were all positive when added to liquefied sputum samples, and the Ct values ​​were not significantly different from those of the control. This indicates that under the experimental conditions of the present invention, the sputum sample matrix treated with the sample processing tube did not significantly interfere with the test results.

[0070] Comparative Example 1 The difference between Comparative Example 1 and Examples 1-4 and Experimental Example 4 is only the difference in primer and probe sequences. The rest are the same. The primer and probe sequences of all comparative examples are from highly cited articles. The primer and probe sequences of Streptococcus pneumoniae are from HKPark etal FEMS MicroboilLett, 2010. Primer and probe sequences for Klebsiella pneumoniae were from Hartman et al JMD, 2009. Primer and probe sequences for Legionella pneumophila from N. Merault etal .2011. Primer and probe sequences for Chlamydia pneumoniae and Mycoplasma pneumoniae were obtained from Kathurman et al . 2011. The primer and probe sequences of Comparative Example 1 are as follows: The primer and probe sequences for Streptococcus pneumoniae SP are: SP2-F: 5'-GCGTTTTAGCAGATAGTGAGATCGA-3' (SEQ ID NO: 16); SP2-R: 5'-TCCCAGTCGGTGCTGTCA-3' (SEQ ID NO: 17); SP2-P: 5'-AAGTGTTACGCAACTGACGAG-3' (SEQ ID NO: 18).

[0071] The primer and probe sequences for Klebsiella pneumoniae KP are: KP2-F: 5'-CGAAAGTGAACGAATTGATGCT-3' (SEQ ID NO: 19); KP2-R: 5'-GTTTCTGCTGCAGATTCGAAGA-3' (SEQ ID NO: 20); KP2-P: 5'-CATCATGCAATAGCCACGT-3' (SEQ ID NO: 21).

[0072] The primer and probe sequences for Legionella pneumophila Lpn are: Lpn2-F: 5'-CAAAGGGCGTTACAGTCAAACC-3' (SEQ ID NO: 22); Lpn2-R: 5'-CAAACACCCCAACCGTAATCA-3' (SEQ ID NO: 23); Lpn2-P:5'-TCTTGGGATTGGGTTGGGTTATTTTAACTCCT-3' (SEQ ID NO:24).

[0073] The primer and probe sequences for Chlamydia pneumoniae CP are: CP2-F: 5'-CGTGGTGCTCGTTATTCTTTACC-3' (SEQ ID NO: 25); CP2-R: 5'-TGGCGAATAGAGAGCACCCAA-3' (SEQ ID NO: 26); CP2-P: 5'-CTTCAACAGAGAAGACCACGACCCGTCA-3' (SEQ ID NO: 27).

[0074] The primer and probe sequences for Mycoplasma pneumoniae (MP) are: MP2-F: 5'-TTTGGTAGCTGGTTACGGGAAT-3' (SEQ ID NO: 28); MP2-R: 5'-GGTCGGCACGAATTTCATATAAG-3' (SEQ ID NO: 29); MP2-P: 5'-TGTACCAGAGCACCCCAGAAGGGCT-3' (SEQ ID NO: 30).

[0075] The comparison of the amplification Ct values ​​of Comparative Example 1 and Experimental Example 4 is shown in Table 9. The amplification results of Comparative Example 1 are as follows: Figure 3As shown in the table, it can be seen that the amplification Ct values ​​of Comparative Example 1 are all delayed compared with the target in the kit, and the amplification fluorescence value of Chlamydia pneumoniae CP in Comparative Example 1 is very low.

[0076] Table 9

[0077] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer-probe combination for on-site detection of pneumonia pathogens, characterized in that: The primer-probe combination includes: primer pairs and probes for detecting sequences of Streptococcus pneumoniae as shown in SEQ ID NOs: 1-3, respectively; primer pairs and probes for detecting sequences of Klebsiella pneumoniae as shown in SEQ ID NOs: 4-6, respectively; primer pairs and probes for detecting sequences of Legionella pneumophila as shown in SEQ ID NOs: 7-9, respectively; primer pairs and probes for detecting sequences of Chlamydia pneumoniae as shown in SEQ ID NOs: 10-12, respectively; and primer pairs and probes for detecting sequences of Mycoplasma pneumoniae as shown in SEQ ID NOs: 13-15, respectively.

2. Use of the primer-probe combination according to claim 1 in the preparation of a product for detecting Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae.

3. The use according to claim 2, characterized in that The products include independent detection reagents, chips and test kits.

4. A kit for on-site detection of pneumonia pathogens, characterized in that: The kit comprises the primer-probe combination according to claim 1.

5. The kit according to claim 4, characterized in that The test kit is an integrated closed microfluidic chip test kit.

6. The kit according to claim 5, characterized in that The integrated closed microfluidic chip kit includes purification reagents and air-dried qPCR reagents.

7. A method for on-site detection of pneumonia pathogens, characterized in that: The method comprises the following steps: (1) preparing the primer-probe combination described in claim 1 into an integrated closed microfluidic kit; (2) adding a sample containing Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, and Mycoplasma pneumoniae to a sample processing tube for processing to obtain a liquid to be tested; (3) dropping the liquid to be tested into the integrated closed microfluidic reagent kit; (4) Place the test kit containing the liquid to be tested on the testing equipment for testing and analysis.

8. The method according to claim 7, characterized in that The step (2) specifically comprises: adding a sample pre-treatment solution and abrasive particles into the sample processing tube; The sample pretreatment solution includes NaOH, NaCl and Triton X-100; When the sample described in step (2) is a sputum sample containing Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, or Mycoplasma pneumoniae, the sputum can be quickly liquefied through a sample processing tube for 1 minute; When the sample described in step (2) is alveolar lavage fluid, oropharyngeal / nasopharyngeal swab preservation fluid, tongue swab preservation fluid, or drainage fluid, it can be directly added to the microfluidic reagent kit sample chamber for detection without being processed through the sample processing tube.

9. The method according to claim 7, characterized in that The step (4) specifically includes: 1) Turn on the rapid detection device; 2) Scan the QR code; 3) Inserting the integrated microfluidic chip kit; 4) Run the test; 5) End and check the test results and amplification curve.

10. Use of the primer-probe combination according to claim 1, the kit according to any one of claims 4 to 6, or the method according to any one of claims 7 to 9 in detecting Streptococcus pneumoniae, Klebsiella pneumoniae, Legionella pneumophila, Chlamydia pneumoniae, or Mycoplasma pneumoniae.

Images