A primer and probe combination, kit and application for detecting multiple respiratory pathogens

Abstract

The present invention discloses a primer and probe combination for detecting multiple respiratory pathogens, characterized in that the primer is an RPA multiplex primer and a nested RPA primer, and the probe is a nested RPA probe; the RPA multiplex primer is specifically a primer pair composed of the upstream or downstream RPA multiplex primers shown in SEQ NO.1-24; the nested RPA primer is specifically a primer pair composed of the upstream or downstream nested RPA primers shown in SEQ NO.25-48; the nested RPA probe is the sequence shown in SEQ NO.49-60. The present invention also discloses a corresponding kit and application. Through the combination of the advantages of several technologies and the uniquely designed primers and probes, 12 respiratory pathogens can be detected in one reaction, and PCR amplification, signal reading, and result reporting can be completed within 1.5 hours; the detection sensitivity is 100 copies / reaction.

Description

Technical Field

[0001] The present invention relates to the field of gene detection, and particularly to a primer and probe combination, a kit and an application for detecting multiple respiratory pathogens. Background Art

[0002] Since the polymerase chain reaction (PCR) technology was invented by Mullis et al. in 1983, it has been widely used in life science research and other aspects. In a traditional PCR reaction, in the presence of a reaction mixture of a DNA template, primers, four dTTPs, and an appropriate buffer, the target DNA fragment is amplified by the catalysis of a DNA polymerase. The PCR reaction generally includes three steps: denaturation, annealing, and extension, and each step is repeated 30 to 40 times, thereby exponentially amplifying a small amount of target DNA fragments to a detectable level. Since the PCR technology can amplify a small number of nucleic acid molecules to a detectable level by the instrument, it has been rapidly applied to multiple fields, such as disease diagnosis, animal and plant pathology research, microbial detection, and so on.

[0003] Based on the powerful amplification ability of PCR, in practical applications, a variety of detection technologies have been developed by combining PCR with other molecular biology methods and immunological methods, etc. For example, fluorescence quantitative PCR technology, multiplex PCR detection technology, etc. have been widely used in disease detection.

[0004] Currently, the gold standard for detecting infectious pathogens is the real-time fluorescence quantitative method based on the polymerase chain reaction (PCR). It requires temperature conversion between cycles, and due to the low amplification efficiency, it takes a long time, it is difficult to detect viral nucleic acids with low copy numbers, resulting in a large ct value, and it is easy to produce false negative results in the medical detection of pathogenic microorganisms, with low sensitivity. At the same time, the disadvantage of this method is that it can only detect 1 kind of pathogenic microorganism at a time. By combining the multiplex PCR technology with the multi-color fluorescence labeling technology, its detection throughput has been improved to a certain extent. However, limited by the types of fluorescence and fluorescence interference with each other, most current products based on multiplex QPCR focus on the detection of 3-5 targets, and the number of pathogens detected in one reaction is small, which is difficult to meet the clinical requirement for multiplex simultaneous detection of pathogens.

[0005] Currently, a variety of isothermal in vitro nucleic acid amplification technologies have been developed, such as TMA technology (transcription-mediated nucleic acid amplification technology), SDA technology (strand displacement nucleic acid amplification technology), LAMP (loop-mediated nucleic acid amplification technology), HDA (helicase-dependent isothermal nucleic acid amplification technology), RPA technology (recombinase-mediated isothermal amplification), and so on. These technologies can achieve efficient nucleic acid amplification at a constant temperature (about 65 degrees or 37 degrees), thus eliminating the need to use a PCR instrument that precisely controls temperature.

[0006] Among them, the RPA technology is recognized as a nucleic acid detection technology that can replace PCR. Its principle is that the recombinase binds to the primer to form a protein-DNA complex, which can search for homologous sequences in double-stranded DNA. Once the primer locates the homologous sequence, a strand exchange reaction will occur to form and initiate DNA synthesis, exponentially amplifying the target region on the template.

[0007] In addition, the RPA technology has multiple tool enzymes for effective amplification. The amplification efficiency is much higher than that of the traditional PCR technology, and the time used is shorter, which can be achieved within 5 minutes at the shortest. Due to the characteristics of short amplification time, no need for expensive instruments, and good specificity of the RPA technology, its application is becoming more and more extensive. The biggest feature of the RPA technology is that only one pair of primers is required to achieve the amplification of the template nucleic acid under the constant temperature condition of about 40 °C, without the need to achieve nucleic acid denaturation and annealing through high and low temperature cycles, so expensive instrument equipment is not required. Moreover, the conventional reaction temperature of 37 °C is easily satisfied, which is suitable for the rapid detection of pathogenic bacteria at the grass-roots level. At present, the RPA technology has been widely used in the detection of viruses in humans, animals or plants.

[0008] However, similarly, the existing isothermal amplification technology can only detect one to several pathogens at a time. At present, most products based on isothermal amplification focus on the detection of 1-4 targets, and the number of pathogens detected in one reaction is small, which is difficult to meet the clinical requirements for multiplex simultaneous detection of pathogens.

[0009] Therefore, the isothermal amplification, multiplex PCR amplification and nested PCR amplification technologies are combined, and specific RPA multiplex primers, nested PCR primers and RPA probes are designed to solve the above problems. Summary of the Invention

[0010] In order to overcome the above-mentioned defects of the prior art, the purpose of the present invention is to provide a primer and probe combination, a kit and an application for detecting multiple respiratory pathogens.

[0011] In order to achieve the purpose of the present invention, the technical solution adopted is:

[0012] A primer and probe combination for detecting multiple respiratory pathogens,

[0013] The primer is an RPA multiplex primer and a nested RPA primer, and the probe is a nested RPA probe;

[0014] The RPA multiplex primer is specifically a primer pair composed of the upstream or downstream RPA multiplex primers shown in SEQ NO.1-24;

[0015] The nested RPA primer is specifically a primer pair composed of the upstream or downstream nested RPA primers shown in SEQ NO.25-48;

[0016] The nested RPA probe is the sequence shown in SEQ NO. 49-60.

[0017] In a preferred embodiment of the present invention, the molar ratio of the upstream or downstream RPA multiple primer in the RPA multiple primer is 1:1.

[0018] During detection, the dosage of each of the above RPA multiple primer mixtures with a concentration of 10 uM is 4 ul.

[0019] During detection, the dosage of each of the above nested RPA primers with a concentration of 10 uM is 2 ul.

[0020] During detection, the dosage of each of the above nested RPA probes with a concentration of 5 pM is 0.6 ul.

[0021] A kit for detecting multiple respiratory pathogens, the kit includes the primer and probe combination, and the multiple respiratory pathogens are coronavirus OC43 type, human metapneumovirus, rhinovirus, influenza A virus type A, influenza A H1N1 virus, influenza B virus, coronavirus HKU1 type, coronavirus NL63 type, coronavirus 229E type, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus.

[0022] A kit for detecting multiple respiratory pathogens, the kit further includes recombinase polymerase lyophilized enzyme powder, hydrolysis buffer solution, magnesium acetate solution and deionized water.

[0023] An application of a kit for detecting multiple respiratory pathogens, the application is to process the sample nucleic acid by isothermal amplification, multiplex PCR amplification and nested PCR amplification for detecting the multiple respiratory pathogens.

[0024] The detection includes the following steps:

[0025] The sample nucleic acid first undergoes a rapid reverse transcription process to convert RNA into cDNA, and then the RPA multiple primer is used to start the first-stage multiplex RPA reaction;

[0026] After the first-stage reaction ends, DNA adsorption magnetic beads are used for purification and dilution of the multiplex RPA amplification product; after the product of the first-stage multiplex RPA is diluted, it is used as the template for the second-stage nested RPA.

[0027] Using the nested RPA primer and the nested RPA probe, in the second-stage nested RPA amplification reaction, the detection and quantification of the target pathogen are achieved by monitoring the change of the fluorescence signal level in each reaction.

[0028] The beneficial effects of the present invention are as follows:

[0029] By combining the advantages of several technologies and using uniquely designed primer probes, 12 respiratory pathogens can be detected in a single reaction, and PCR amplification, signal reading, and result reporting can be completed within 1.5 hours. The detection sensitivity is 100 copies / reaction. Description of the Drawings

[0030] Figure 1 This is the result diagram for detecting the plasmid standard of coronavirus OC43 type (HCoVOC43) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0031] Figure 2 This is the result diagram for detecting the plasmid standard of human metapneumovirus (HMPV) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0032] Figure 3 This is the result diagram for detecting the plasmid standard of rhinovirus (RhV) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0033] Figure 4 This is the result diagram for detecting the plasmid standard of influenza A virus type A (FluA) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0034] Figure 5 This is the result diagram for detecting the plasmid standard of influenza A virus H1N1 (2009H1N1) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0035] Figure 6 This is the result diagram for detecting the plasmid standard of influenza B virus (FluB) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0036] Figure 7 This is the result diagram for detecting the plasmid standard of coronavirus HKU1 type (HCoVHKU1) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0037] Figure 8 This is the result diagram for detecting the plasmid standard of coronavirus NL63 type (HCoVNL63) in Example 1 of the present invention. The results show that both 100 copies replicates are positive.

[0038] Figure 9This is the result diagram for detecting the plasmid standard of coronavirus type 229E (HCoV229E) in Example 1 of the present invention. The results show that both 100 copies of the duplicate wells are positive.

[0039] Figure 10 This is the result diagram for detecting the plasmid standard of parainfluenza virus type 2 (PIFV2) in Example 1 of the present invention. The results show that both 100 copies of the duplicate wells are positive.

[0040] Figure 11 This is the result diagram for detecting the plasmid standard of parainfluenza virus type 3 (PIFV3) in Example 1 of the present invention. The results show that both 100 copies of the duplicate wells are positive.

[0041] Figure 12 This is the result diagram for detecting the plasmid standard of respiratory syncytial virus in Example 1 of the present invention. The results show that both 100 copies of the duplicate wells are positive. Detailed implementation mode

[0042] A primer and probe combination for detecting multiple respiratory pathogens,

[0043] The primers are RPA multiplex primers and nested RPA primers, and the probes are nested RPA probes;

[0044] The RPA multiplex primers are specifically primer pairs composed of the upstream or downstream RPA multiplex primers shown in SEQ NO.1-24;

[0045] The nested RPA primers are specifically primer pairs composed of the upstream or downstream nested RPA primers shown in SEQ NO.25-48;

[0046] The nested RPA probe is the sequence shown in SEQ NO.49-60.

[0047] The molar ratio of the upstream or downstream RPA multiplex primers in the RPA multiplex primers is 1:1.

[0048] During detection, the dosage of the above-mentioned RPA multiplex primer mixture with a concentration of 10 uM is 4 ul.

[0049] During detection, the dosage of the above-mentioned nested RPA primers with a concentration of 10 uM is 2 ul.

[0050] During detection, the dosage of the above-mentioned nested RPA probes with a concentration of 5 pM is 0.6 ul.

[0051] The corresponding relationship between the pathogen and the primer and probe, as well as the primer and probe sequences are as described in Table 1.

[0052] Table 1

[0053]

[0054]

[0055]

[0056]

[0057]

[0058] Note:

[0059] * In the name of the primer-probe, "F" represents the upstream primer, "R" represents the downstream primer, and "P" represents the probe.

[0060] The downstream primer of the probe is coupled with biotin at the 5'-end, FAM is coupled to the upstream of the probe, a base is replaced by THF (tetrahydrofuran) at the middle position, and phosphorylation treatment is performed at the 3'-end.

[0061] The primer set and the probe are both purified by PAGE and combined with mass spectrometry to test the synthesized product.

[0062] The detection process using the kit of the present invention includes the following steps:

[0063] 2) Sample treatment and nucleic acid extraction to obtain the nucleic acid template of the sample to be tested;

[0064] 2) Prepare the first-stage RPA multiplex amplification reagent and perform multiplex amplification using the obtained nucleic acid as a template; specifically: add 12.5 μL of Buffer A Buffer, 4 μL of 10 μM RPA multiplex primer mixture, 8.8 μL of sterile double-distilled water, and 1 μL of template to the reaction unit tube containing the dry powder of the RPA nucleic acid amplification basic reagent. To ensure that the reaction proceeds simultaneously, 1.25 μL of BBuffer (MgAc) should be added to the lid of the eight-well tube, carefully flip and cover it, fully mix the RPA amplification system, centrifuge at 5,000×g for 10 s, and place it on a 40°C PCR instrument for reaction for 30 min.

[0065] The specific components of Buffer A are: sodium chloride, Tris-HCL buffer, potassium chloride, magnesium chloride, dithiothreitol or polyvinylpyrrolidone.

[0066] 3) Use DNA adsorption magnetic beads to purify and dilute the multiplex RPA amplification product.

[0067] 4) Configure the second-stage nested RPA amplification reagent, perform nested RPA amplification using the purified product obtained as a template, and complete fluorescence detection; specifically: Add 12.5 μL of Buffer A Buffer, 1 μL of 10 μM upstream primer, 1 μL of 10 μM downstream primer, 0.6 μL of probe, 8 μL of sterile double-distilled water, and 1 μL of template to the reaction unit tube containing the dry powder of the RPA nucleic acid amplification fluorescent reagent. To ensure simultaneous reaction, 1.25 μL of B Buffer (MgAc) should be added to the lid of the eight-well strip tube, carefully flip and cover it, mix the RPA amplification system thoroughly, centrifuge at 5,000×g for 10 s, place it on the QPCR instrument for reaction for 30 min, set the QPCR instrument to 40 °C for 3 min, 40 °C for 45 s / cycle, 40 cycles, and detect the fluorescence in the FAM channel.

[0068] 5) Use a fluorescent probe to detect the amplification product. Compare with the negative control. If an S-shaped curve is determined in the detection well on the QPCR instrument, it is judged as positive, indicating that the sample contains the corresponding pathogen; if no S-shaped curve is seen, it is judged as negative, indicating that the sample does not contain the corresponding pathogen.

[0069] The following combines specific embodiments to further explain and illustrate the present invention:

[0070] Example 1 Detection of plasmid standards of respiratory pathogens to verify the sensitivity of the method:

[0071] 1. Preparation and dilution of plasmid standards:

[0072] By performing sequence alignment and analysis on the nucleic acid sequences related to respiratory pathogens reported in the Genebank database and published domestic and foreign literature, select the highly conserved segments without secondary structure as the amplification target fragments. For the 12 selected pathogens, prepare their plasmid standards with a plasmid size of about 400 bp; the synthesized stock solution of the plasmid standard is 10 10 copies / μl, and dilute it to 100 copies / μl using TE solution according to a gradient. The sensitivity of conventional QPCR detection is 100 copies / reaction.

[0073] 2. First-stage multiplex RPA amplification:

[0074] Use the basic nucleic acid amplification reagent of Hangzhou Zhongce Biotechnology Co., Ltd. to prepare the first-stage system.

[0075] Configure the reaction system according to the components listed in Table 2 (the reaction solution is prepared on ice), shake and mix well, and centrifuge briefly.

[0076] Table 2 Configuration of the first-stage amplification system

[0077]

[0078] To ensure that the reactions proceed simultaneously, 1.25 μL of B Buffer (MgAc) should be added to the lid of the 8-strip tube. Carefully flip the lid to cover the tube and thoroughly mix the RPA amplification system. Centrifuge at 5,000×g for 10 s and place it in a 40°C PCR instrument for a 30-min reaction.

[0079] 3. Purification of the first-stage amplification products:

[0080] Use VAHTS DNA Clean Beads from Nanjing Novoprotein Scientific Inc. to adsorb magnetic beads for the purification and dilution of multiplex RPA amplification products. Pipette 30 μL of magnetic beads into the PCR tube from the previous amplification step (total volume 61.25 μL), mix well by shaking, and let it stand for 5 min. Place the PCR tube on a magnetic stand and adsorb for 2 - 3 min. After the solution becomes clear, discard the supernatant. Add 100 μL of 80% ethanol (total volume 100 μL), let it stand for 30 s, discard the supernatant, repeat the washing once, and thoroughly discard the supernatant. Add 100 μL of deionized water, mix well by shaking, place the PCR tube on the magnetic stand, and when the solution becomes clear, pipette the supernatant and transfer it to a new centrifuge tube. Then add 100 μL of NF water to dilute to a total volume of 200 μL.

[0081] 4. Second-stage nested RPA amplification:

[0082] Use the fluorescent nucleic acid amplification reagent from Hangzhou Zhongce Biotechnology Co., Ltd. to prepare the second-stage reaction system.

[0083] Prepare the reaction system according to the components listed in Table 3 (the reaction solution preparation is carried out on ice), mix well by shaking, and centrifuge briefly. Among them, two replicates are set for the detection of each plasmid standard, and a negative control is added.

[0084] Table 3 Configuration of the second-stage amplification system

[0085]

[0086]

[0087] To ensure that the reactions proceed simultaneously, 1.25 μL of B Buffer (MgAc) should be added to the lid of the 8-strip tube. Carefully flip the lid to cover the tube and thoroughly mix the RPA amplification system. Centrifuge at 5,000×g for 10 s and place it in a QPCR instrument for a 30-min reaction. The QPCR instrument is set at 40°C for 3 min, 40°C for 45 s / Cycles, 40 Cycles, and the fluorescence of the FAM channel is detected.

[0088] 5. Result analysis:

[0089] After amplification, the data is processed by the software built into the QPCR instrument. Compared with the negative control, if an S-shaped curve is detected in the test well on the QPCR instrument, it is determined as positive; if no S-shaped curve is seen, it is determined as negative.

[0090] 6. Detection results of plasmid standards:

[0091] The prepared plasmid standards of 100 copies were used for detection, and the results are shown in Table 4.

[0092] Table 4

[0093]

[0094]

[0095] The experimental results of each pathogen plasmid standard are as Figures 1 - 12 shown.

[0096] In summary, the main innovation points of the present invention are as follows:

[0097] 1. Compared with traditional PCR and fluorescence quantitative PCR, the operation of the present invention is simpler, requires less time, and has low requirements for instruments.

[0098] 2. The present invention can detect 12 respiratory pathogens through a single reaction, greatly improving the detection efficiency, providing more comprehensive result references for doctors, and meeting clinical needs.

[0099] 3. The present invention can achieve a detection sensitivity of 100 copies / reaction, which is consistent with the sensitivity of the existing gold standard QPCR detection, ensuring the detection accuracy. Sequence Listing <110> Shanghai Shiouwen Gene Technology Co., Ltd. <120> A primer and probe combination, kit and application for detecting multiple respiratory pathogens <130> 20220524 <160> 60 <170> SIPOSequenceListing 1.0 <210> 1 <211> 33 <212> DNA <213> homo sapiens <400> 1 caaccctagt agttcagagg tggatatgat ttg 33 <210> 2 <211> 33 <212> DNA <213> homo sapiens <400> 2 caaagcatct tgaagtagca cctccaaata ata 33 <210> 25 <211> 33 <212> DNA <213> homo sapiens <400> 25 caaccctagt agttcagagg tggatatgat ttg 33 <210> 26 <211> 33 <212> DNA <213> homo sapiens <400> 26 atttcacgta ttaggctaga ctgcacacaa cac 33 <210> 49 <211> 49 <212> DNA <213> homo sapiens <400> 49 aatttgtcct gaaaatcatg tgatggtgga ttgtcgccga cttcttaaa 49 <210> 3 <211> 33 <212> DNA <213> homo sapiens <400> 3 tattgtcaga atgcagggtc aactgtctac tac 33 <210> 4 <211> 32 <212> DNA <213> homo sapiens <400> 4 ccaatggaac agcttactcc tttgtaacaa gc 32 <210> 27 <211> 30 <212> DNA <213> homo sapiens <400> 27 aattaatgtt gctgagcaat caaaggagtg 30 <210> 28 <211> 30 <212> DNA <213> homo sapiens <400> 28 cttactcctt tgtaacaagc aaccagagcc 30 <210> 50 <211> 49 <212> DNA <213> homo sapiens <400> 50 ttacccatgc aaagtcagca caggaaggca tcctatcagt atggttgca 49 <210> 5 <211> 33 <212> DNA <213> homo sapiens <400> 5 aactacgtaa tggctagtaa caccattgat tta 33 <210> 6 <211> 31 <212> DNA <213> homo sapiens <400> 6 ctcattacga cctcaacaac attggattat g 31 <210> 29 <211> 33 <212> DNA <213> homo sapiens <400> 29 agtttaatga catggtgcga agacttgcat gtg 33 <210> 30 <211> 31 <212> DNA <213> homo sapiens <400> 30 ctcattacga cctcaacaac attggattat g 31 <210> 51 <211> 50 <212> DNA <213> homo sapiens <400> 51 catgtgcttg gttgagactc ctccggcccc tgaatgcggc taaccttaac 50 <210> 7 <211> 33 <212> DNA <213> homo sapiens <400> 7 tagatattga aagatgagtc ttctaaccga ggt 33 <210> 8 <211> 33 <212> DNA <213> homo sapiens <400> 8 atgttatctc tcttttcagc ttcctgtaca gtt 33 <210> 31 <211> 32 <212> DNA <213> homo sapiens <400> 31 aagatgtctt tgcagggaag aacactgatc tc 32 <210> 32 <211> 29 <212> DNA <213> homo sapiens <400> 32 atgttgtttg ggtctccatt tccatttag 29 <210> 52 <211> 50 <212> DNA <213> homo sapiens <400> 52 catgtgcttg gttgagactc ctccggcccc tgaatgcggc taaccttaac 50 <210> 9 <211> 33 <212> DNA <213> homo sapiens <400> 9 atttcatcaa ttatgaggag ctaagagagc aat 33 <210> 10 <211> 33 <212> DNA <213> homo sapiens <400> 10 gacttctttc cctttatcat taatgtagga ttg 33 <210> 33 <211> 33 <212> DNA <213> homo sapiens <400> 33 ctcagtgtca tcatttgaaa ggtttgagat att 33 <210> 34 <211> 33 <212> DNA <213> homo sapiens <400> 34 gacttctttc cctttatcat taatgtagga ttg 33 <210> 53 <211> 49 <212> DNA <213> homo sapiens <400> 53 acgctggagc aaaaagcttc tacaaaaatt taatatggct agttaaaaa 49 <210> 11 <211> 33 <212> DNA <213> homo sapiens <400> 11 atgcttaact gatatacaaa aagcactaat tgg 33 <210> 12 <211> 33 <212> DNA <213> homo sapiens <400> 12 attcaggtac atgaccatga gacagtatag tag 33 <210> 35 <211> 33 <212> DNA <213> homo sapiens <400> 35 aagaaaaaga agattcatca cagagccctt atc 33 <210> 36 <211> 33 <212> DNA <213> homo sapiens <400> 36 tacatgacca tgagacagta tagtagcgct gag 33 <210> 54 <211> 49 <212> DNA <213> homo sapiens <400> 54 tgagagaaaa atgagaagat gtgtgagctt tcatgaagca tttgaaata 49 <210> 13 <211> 33 <212> DNA <213> homo sapiens <400> 13 ttatccatgc ttgtgagtgt ggtttaatca taa 33 <210> 14 <211> 32 <212> DNA <213> homo sapiens <400> 14 attatcataa acaaatccaa cactttccat cg 32 <210> 37 <211> 33 <212> DNA <213> homo sapiens <400> 37 tattttactt tccacatttt tcatctctct gcc 33 <210> 38 <211> 30 <212> DNA <213> homo sapiens <400> 38 cccacttgaa gccgagaccg tatttgctgg 30 <210> 55 <211> 50 <212> DNA <213> homo sapiens <400> 55 acgtgttggt tgtcctcagc gtccctccca taggtcgcaa tgattaaaac 50 <210> 15 <211> 33 <212> DNA <213> homo sapiens <400> 15 tacttcttgt tgatggccat aagattgcta ctc 33 <210> 16 <211> 33 <212> DNA <213> homo sapiens <400> 16 atatggtgcc ttatcagaac taaccaaaag agg 33 <210> 39 <211> 33 <212> DNA <213> homo sapiens <400> 39 ttgctacacc tagtaccaca attgtttgtg acc 33 <210> 40 <211> 33 <212> DNA <213> homo sapiens <400> 40 aactaaccaa aagaggcatg taaaatgaag gag 33 <210> 56 <211> 49 <212> DNA <213> homo sapiens <400> 56 aagccagact ggttgggcat tctacgtccg tgctaaacat ggtgatttt 49 <210> 17 <211> 33 <212> DNA <213> homo sapiens <400> 17 tctattatct tggcacagga cctcataaag atg 33 <210> 18 <211> 33 <212> DNA <213> homo sapiens <400> 18 tttcctgagg cttgtcaaaa cctaaagatt taa 33 <210> 41 <211> 33 <212> DNA <213> homo sapiens <400> 41 aattcagaac cagagatacc acacttcaat caa 33 <210> 42 <211> 33 <212> DNA <213> homo sapiens <400> 42 attccgagat tgagatttgg attcaacacg act 33 <210> 57 <211> 49 <212> DNA <213> homo sapiens <400> 57 tgttactgtt gctgaagaac ctgactcccg tgctccttcc cgttctcag 49 <210> 19 <211> 33 <212> DNA <213> homo sapiens <400> 19 cgctccttta caggatcaat acagattttt cta 33 <210> 20 <211> 32 <212> DNA <213> homo sapiens <400> 20 tatttaagag ggagttagcc gatgcattgt ag 32 <210> 43 <211> 33 <212> DNA <213> homo sapiens <400> 43 atacagattt ttctaaagga attcctccga tca 33 <210> 44 <211> 33 <212> DNA <213> homo sapiens <400> 44 gcattacgtg acatgagttc tggattatta agc 33 <210> 58 <211> 49 <212> DNA <213> homo sapiens <400> 58 tcctggagtc atgccatgca atgcaacaag tttttgccct gctaattgc 49 <210> 21 <211> 33 <212> DNA <213> homo sapiens <400> 21 aagaataaaa tggacatggc ataatgtgct atc 33 <210> 22 <211> 33 <212> DNA <213> homo sapiens <400> 22 tcgtttaccc tttcagttga tgttgagtaa gtt 33 <210> 45 <211> 33 <212> DNA <213> homo sapiens <400> 45 ataatgtgct atcaagacca ggaaacaatg aat 33 <210> 46 <211> 33 <212> DNA <213> homo sapiens <400> 46 atttttgcga gtctaatatg acagatgaca caa 33 <210> 59 <211> 48 <212> DNA <213> homo sapiens <400> 59 tgtccatggg gacattcatg cccagatgga tgtataacag gagtatat 48 <210> 23 <211> 33 <212> DNA <213> homo sapiens <400> 23 caagcttaac aactgaaatt caaatcaaca ttg 33 <210> 24 <211> 33 <212> DNA <213> homo sapiens <400> 24 cttcatagaa gctgttggct atatccttgg gta 33 <210> 47 <211> 32 <212> DNA <213> homo sapiens <400> 47 catgactctc ctgattgtgg gatgataata tt 32 <210> 48 <211> 33 <212> DNA <213> homo sapiens <400> 48 aagctgttgg ctatatcctt gggtaataaa cct 33 <210> 60 <211> 49 <212> DNA <213> homo sapiens <400> 60 aaccaaatta gcagcaggag atagatcagg tcttacagct gtgattagg 49

Claims

1. A primer and probe combination for detecting multiple respiratory pathogens, characterized in that, the primers are RPA multiplex primers and nested RPA primers, and the probes are nested RPA probes; the RPA multiplex primers are specifically primer pairs composed of the upstream and downstream RPA multiplex primers shown in SEQ NO.1-24; the nested RPA primers are specifically primer pairs composed of the upstream and downstream nested RPA primers shown in SEQ NO.25-48; the nested RPA probe is the sequence shown in SEQ NO.49-60.

2. The primer and probe combination for detecting multiple respiratory pathogens according to claim 1, characterized in that, the molar ratio of the upstream or downstream RPA multiplex primer in the RPA multiplex primers is 1:

1.

3. A kit for detecting multiple respiratory pathogens, the kit comprising the primer and probe combination according to claim 1 or 2, characterized in that, the multiple respiratory pathogens are coronavirus OC43 type, human metapneumovirus, rhinovirus, influenza A virus type A, influenza A H1N1 virus, influenza B virus, coronavirus HKU1 type, coronavirus NL63 type, coronavirus 229E type, parainfluenza virus type 2, parainfluenza virus type 3, respiratory syncytial virus.

4. A kit for detecting multiple respiratory pathogens according to claim 3, characterized in that, the kit further comprises recombinase polymerase freeze-dried enzyme powder, hydrolysis buffer solution, magnesium acetate solution and deionized water.

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