A primer combination for diagnosing multiple respiratory pathogens and its application

Abstract

The present invention belongs to the field of gene detection, and particularly relates to a primer combination for diagnosing multiple respiratory pathogens and its application. The present invention provides a primer combination, which includes specific primer sequences: SEQ ID NO: 1 - SEQ ID NO: 18, and has the advantages of high detection sensitivity and good specificity for low-concentration pathogens. The present invention uses the SARSeq technology, combines with the primer combination, enriches the detection fragments of multiple pathogens through PCR amplification and determines the nature by the next-generation sequencing method. Compared with the traditional fluorescence quantitative PCR detection, it has the advantages of high detection specificity and the detection data of each pathogen are not easily interfered with each other. The technical solution of the present invention has the advantages of simultaneous detection of multiple pathogens and low single-sample detection cost, without further constructing a sequencing library and with relatively low subsequent sequencing data volume requirements.

Description

Technical Field

[0001] The present invention belongs to the field of gene detection, and particularly relates to a primer combination for diagnosing multiple respiratory pathogens and its application. Background Art

[0002] Acute respiratory infections are a group of diseases mainly manifested as respiratory system diseases caused by various viruses, bacteria, mycoplasmas and chlamydiae, including acute upper respiratory tract infections and acute lower respiratory tract infections. [1] Acute respiratory infections are one of the most common infectious diseases in humans worldwide, with the highest incidence among various infectious diseases. [2] According to the data provided by the World Health Organization, the number of deaths caused by acute respiratory infections worldwide in 2016 reached as high as three million, with a mortality rate of four per ten thousand. [3] According to clinical research data, among the domestic population with acute respiratory infections, more than 34.8% of the patients are caused by viral infections, and more than 22.8% of the patients are caused by bacterial infections. [4] .

[0003] Conventional fluorescence quantitative PCR (Real-Time PCR) is favored and widely used in the detection of respiratory pathogens due to its characteristics such as rapidity, low cost and high sensitivity. However, problems such as its weak multiplex detection ability and low detection specificity still limit its application in clinical diagnostic detection. In recent years, the applications of mNGS (metagenomic next-generation sequencing) and tNGS (pathogen-targeted high-throughput sequencing) have effectively expanded the detection range of respiratory pathogens. Their characteristics such as strong multiplex detection ability and high specificity well complement the current detection needs. However, reasons such as the high technical difficulty, long detection time, high single-sample detection cost and unsuitability for large-scale detection of these sequencing methods also result in less clinical application of such sequencing methods. Based on the fact that there are still many defects and deficiencies in current large-scale detection technologies in terms of detection diversity, detection specificity and detection cost control, it is still crucial to develop more accurate, rapid and low-cost large-scale detection methods.

[0004] SARSeq (saliva analysis by RNA sequencing) technology is a newly developed and applied detection method in the near term. It has advantages such as low cost, good sensitivity and high accuracy, and can be applied to large-scale rapid detection of SARS-CoV-2 and multi-pathogen detection. [5] SARSeq technology with high throughput, high sensitivity and specificity is very suitable for regular monitoring of infections in large tissues or populations. In addition, SARSeq technology can be used in epidemiological studies to understand the transmission dynamics of infections and investigate the interactions between different pathogens in the population.

[0005] Primers are crucial for the specificity of PCR reactions. The specificity of PCR requires primers to specifically bind to the target gene and not to other non-target genes. The sensitivity of PCR requires the polymerase to effectively extend the primers. It can be seen that the quality of primer design is closely related to the PCR results. During the process of using software for primer design, there are multiple primer selections. However, each primer has certain defects. During the process of software primer selection, some artificial modifications can be made and combined with primer analysis software to obtain optimized primers.

[0006] [1] Bellos A, MulhollAnd K, O'Brien KL, QAzi SA, Gayer M, CheCChi F. The burden of ACuTe respirATory infeCTions in Crisis-AffeCTed populATions: A sysTemATiC review. Confl HeAlTh. 2010;4:3.

[0007] [2] GBD 2019 DiseAses And Injuries CollAborATors. GlobAl burden of 369 diseAses And injuries in 204 CounTries And TerriTories, 1990 - 2019: A sysTemATiC AnAlysis for The GlobAl Burden of DiseAse STudy 2019 [published CorreCTion AppeArs in LAnCeT. 2020 Nov 14;396(10262):1562]. LAnCeT. 2020;396(10258):1204 - 1222.

[0008] [3] World HeAlTh OrGAnizATion. "GlobAl heAlTh esTimATes 2016: deAThs by CAuse, AGe, sex, by CounTry And by reGion, 2000–2016." GenevA: World HeAlTh OrGAnizATion(2018).

[0009] [4]Li ZJ,Zhang HY,Ren LL,et al.Etiological and epidemiological features of acute respiratory infections in China.Nat Commun.2021;12(1):5026.

[0010] [5]Yelagandula,Ramesh,et al."SARSeq,A robust and highly multiplexed NGS assay for parallel detection of SARS-CoV2 and other respiratory infections."medRxiv(2020). Summary of the Invention

[0011] To solve the above problems,the present invention provides a primer combination,which includes specific primer sequences:SEQ ID NO:1 - SEQ ID NO:18,and has the advantage of high detection sensitivity for low-concentration pathogens.This patent uses the SARSeq technical method,in combination with the primer combination,to enrich the detection fragments of multiple pathogens through PCR amplification.Compared with the traditional fluorescence quantitative PCR for detecting one or more pathogens,it has the advantages of high detection specificity and non-interference of detection data of each pathogen with each other.The primer combination and its application provided by this patent have the advantage of low single-sample detection cost.The multiplex primer combination diagnostic technology using the SARSeq technology does not require further construction of a sequencing library and has a lower requirement for subsequent sequencing data volume,resulting in an obvious single-sample detection cost advantage.

[0012] The present invention provides a primer combination,including specific primer sequences;the specific primer sequences consist of SEQ ID NO:1 - SEQ ID NO:18,as shown in the following table:

[0013]

[0014] Note:In the sequence,W:(A / T);Y:(C / T);R:(A / G);GAPDH(glyceraldehyde-3-phosphate dehydrogenase) is used as an internal reference and quality control for detection,and its detection value can show the success of sampling and the success of the entire reaction system.

[0015] Specifically,the primer combination further includes an adapter sequence and an Index.

[0016] More specifically,the adapter sequence is SEQ ID NO:19 or SEQ ID NO:20.

[0017] Specifically, 1-4 random bases are added upstream of the Index, and the random bases are represented by N.

[0018] More specifically, the forward sequences of the said Index can be selected from: NCCAATACT, NNTCGCGCAT, NNNGTGTGAAC, NNNNGGTGGCAC, NTAGGTGCT, NNGAGCGGTT, NNNAGGATTAG, NNNNGTGAGCCA, NAATTATGC, NNTTACGCCG, NNNGTAGTGAT, NNNNTGGTTGCA, NGTCCGACC, NNGCCAGCCG, NNNCTCGTGTC, NNNNGCCGTGGC, NGTAGCCAC, NNGACTTATA, NNNGAACGTCG, NNNNATGCGTAG, NAATTAGAT, NNTTACGATA, NNNCTGTACAA, NNNNCGTGGTTG, NAATAACGT, NNTTCTTGAA, NNNGGCAGATC, NNNNCTATGTTA, NGTTGACGC, NNATCTACGA, NNNCTCGACAG, NNNNGAGGCTGC, NCCTCGTAG, NNCATAGGCA, NNNAGATGAAC, NNNNCCGAGTAT, NAATATTGA, NNGTATACCG, NNNGATCCAAC, NNNNAGATACGC, NGGTATCTT, NNCCTCTGGC, NCCATTGTG, NNACTACGGT, NNNAAGTGCTA, NNNNGCCGAACG, NTGTCCACG, NNGACACACT, NNNAATATGCT, NNNNTTCTCATA, NTCTGTGAT, NNCCGAACTT, NNNGTCTAACA, NNNNGACGCCAT, NGCCAATGT, NNCCAACGTC, NNNGTAGATAA, NNNNCTTACGGC, NCCAAGTGC, NNCTAACTCA, NNNAATATCTG, NNNNTTATATCA, NCTGCGGAT, NNGCGGCTTG, NNNGAGTTGAT, NNNNGCACTGAG, NGACCACCT, NAATAGCAA, NCCTACCGG, NNGGAGGATG, NNNCGCTGAAT, NNNNTGTGACGA, NAATAGATT, NNTTAGCGCA, NNNGCGGCCGT, NNNNCAGTAACC, NGCCTAGTA, NNCACGGCGC, NNNGGTGCAGA, NNNNTCGCTGAC, NCAGCCAGT, NNCGTCAACC, NGCCGGCGA, NNGCCTCCGG, NNNAATAGTCC,NNNNTTAGACGT, NGTGGACTA, NNCACGGACG, NNNCACTAGAG, NNNNGCAGATG, NCTCTCACG, NNGGAATCAC, NNNCGTTGACG, NNNNCATCAGGT, NCGTTGTAA, NNGGCACGGT; The reverse sequences of the Index can be selected from: AGCTTGGT, AGCTTATG, ATAACGAC, GATGGAGT, TGATCTAA, AGCTATAT, AGCAGAGA, GACGTAAG, AGGCCTGA, TAAGATTA, AGGCGAAT, TTGTTCTT, TACTCCTG, GACTGGCG, ATATATAC, TAGATCGG, AGGCAAGC, TCCAGGTA, AGGTCAAG, TACACTGG, TCCTTGCG, AGGACCTC, TAAGCATT, AGGATATT, AATCGTTA, GTCTACAT, CGCTGCTC, GATCAACA, CGAAGGAC, GATGCCGG, CTACGAAG, GATGCGTC, CTACGGCA, GATTCCTT, CTACTCGA, GATTCGAG, AATCGGCG, TTCGCCGA, CTGGCCTC, GAACTTAT, CGTATTGG, GAAGCACA, CTTAATAC, GAAGTCTT, GAAGAGGC, CGGATAAC, GAATCTGG, CTGATTGA, AATCCGTT, TGCGTACA, GAATCAAT, TGAGTCAG, GAATGCTC, GAATATCC, CTTATGAA, TCGGCACC, AAGAAGCG, CTACGAT, TCGGTCGA, TCGGTAAG, AAGATACA, GTCGCTGT, TCGGATGT, CGAGCCGG, CGATTATC, TCGAAGCT, CTATCATT, AAGTTATC, CGAACGGA, CTACTGAC, TCTTAAGT, TTAGAGTC, AAGACGAA, TTATTATG, CGCTATTA, TCTATCAG, CGGTGGTA, TCACCAAT, CTGGAAGC, CGTAAGAG, AAGAGAGC, TCAACGAG, TGCGAGAC, CCTGGTGT, AAGTAAGT, TGACTGAA, AAGACTGT, CAATGATG, CACAGTAA, TGGTCATT, CAACCGTG, TGGTGCAC, CCACAATG, TGTGTGCC, CACCACGG,TGTGTTAA。,

[0019] On the other hand, the present invention provides a kit for simultaneously detecting multiple respiratory pathogens, and the kit includes the aforementioned primer combination.

[0020] Specifically, the kit further includes, but is not limited to, sample extraction solution, buffer solution, enzyme, dNTPs.

[0021] Specifically, the final concentration of each primer in the primer combination is 0.2 - 1 μM.

[0022] Preferably, the final concentration of each primer in the primer combination is 0.2 - 0.5 μM.

[0023] More preferably, the final concentration of each primer in the primer combination is 0.25 μM.

[0024] Specifically, the respiratory pathogens may be one or more of influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, novel coronavirus, rhinovirus, and Mycoplasma pneumoniae.

[0025] Specifically, the detection method of the kit includes:

[0026] (1) Reverse transcription;

[0027] (2) Performing the first round of PCR on the reverse transcription product in step (1) using the aforementioned primer combination or kit;

[0028] (3) Mixing the PCR products in step (2) and removing the excess primers;

[0029] (4) Performing the second round of PCR on the PCR product in step (3) using the reported primers;

[0030] (5) Mixing the products in step (4) and purifying the products;

[0031] (6) Sequencing the purified product of the second round of PCR in step (5);

[0032] (7) Analyzing the sequencing results in step (6).

[0033] More specifically, the extraction method of the sample in the reverse transcription system may be extraction using a kit, and the kit is a DNA or RNA rapid extraction kit, and the extraction method includes mixing and incubation.

[0034] The incubation temperature is 92 - 98 °C, preferably 95 °C, and the incubation time is 5 minutes.

[0035] The reverse transcription reaction system is shown in the following table:

[0036] Component Added Volume (μL) Nuclease-Free Water 9-10 10×PCR Top buffer A 2-3 dNTP 0.8-1.5 Reverse Transcription Random Primer 0.1-0.5 DTT 0.1-0.2 RNase Inhibitor 0.2-0.8 High-Temperature Reverse Transcriptase 0.2-0.5 Sample 5-15

[0037] The reverse transcription reaction conditions are set as shown in the following table:

[0038] Temperature Time Number of Cycles 20-30℃ 5 minutes 1 35-40℃ 45 minutes 1 80-90℃ 5 seconds 1 10-15℃ /

[0039] In the primer combination of the first-round PCR reaction system, the concentration of each primer is 2.5 μM.

[0040] The system used for the first-round PCR is as shown in the following table:

[0041]

[0042]

[0043] The reaction conditions for the first-round PCR are set as shown in the following table:

[0044]

[0045] The annealing temperature of the first-round PCR reaction is preferably 58 °C.

[0046] The forward sequence composition of the PCR2 primer used in the second-round PCR can be: sequencing adapter sequence

[0047] +index+upstream adapter sequence of the PCR1 product =

[0048] AATGATACGGCGACCACCGAGATCTACAC+Index+ACACTCTTTCCCTACACGACGCTCTTCCGATCT;

[0049] The relevant index sequences can be selected from: AATAACGT, CCTCGTAG, GGTATCTT, AATATGCT, GTAGATAA, GAGTTGAT, AATAGATT, CAGCCAGT, CACTAGAG, AATAGCAA, GAGAACAA, CCAGTGGT, TTCTTGAA, CATAGGCACCTCTGGC, TTCTCATA, CTTACGGC, GCACTGAG, TTAGCGCA, CGTCAACC, GCAGATGG, TGATCGGT, TGTGAATC;

[0050] The reverse sequence composition of the PCR2 primer can be: sequencing adapter sequence+index+downstream adapter sequence of the PCR1 product =

[0051] CAAGCAGAAGACGGCATACGAGAT+Index+GTGACTGGAGTTCAGACG TGTGCTCTTCCGATCT

[0052] The relevant Index sequences can be selected from: AATCGTTA, CTACGGCA, CGTATTGG, AATCCGTT, AAGAAGCG, CGATTATC, AAGACGAA, AAGAGAGC, CACAGTAA, AAGTTATC, TGAACAGG, TTCTGGTG, GTCTACAT, GATTCCTT, GAAGCACA, TGCGTACA, CTCACGAT, TCGAAGCT, TTATTATG, TCAACGAG, TGGTCATT, GTACAGCT, CAGTGTGG, CAATCGAA, CGCTGCTC, CTACTCGA, CTTAATAC, GAATCAAT, TCGGTCGA, CTATCATT, CGCTATTA, TGCGAGAC, CAACCGTG, CAACTGCT, TTCCACCA, AAGTACAG.

[0053] Purification of the first-round PCR products: Taking each 96-well plate as a whole, collect 5 μL of the first-round PCR products from each well and mix them (final volume: 96 wells (this number of wells can be reduced according to the number of test samples) × 5 μL / well), and mix well and centrifuge.

[0054] Add 2 μL of illustra ExoProStar 1-step to the 5 μL of PCR mixture from the previous step, mix well and centrifuge. The purification conditions are set as shown in the following table:

[0055] Temperature Time Number of Cycles 37℃ 30 minutes 1 80℃ 15 minutes 1 12℃ /

[0056] The reaction system for the second-round PCR is as shown in the following table:

[0057] Component Added Volume (μL) Nuclease-Free Water 35-40 10×PCR Top bufferE 3-8 dNTP 1-1.5 dUTP 0.5-0.8 Ultra-Sensitive High-Tolerance Hot Start Taq DNA Polymerase 0.3-0.7 PCR2 Primer 1-3 PCR Product after the First Round of Purification 2-3

[0058] The reaction conditions for the second-round PCR are set as shown in the following table:

[0059]

[0060]

[0061] Collect the second-round PCR products and mix 5 - 20 μL / well. Take 20 μL of the final mixture for the next step. Centrifuge and purify according to Gel Extraction Kit Vazyme DC301-1, and the purified DNA products are used for sequencing.

[0062] In another aspect, the present invention also provides the use of the aforementioned primer combination or the aforementioned kit in food detection.

[0063] Technical effects achieved by the present invention: The present invention provides a primer combination, which includes specific primer sequences: SEQ ID NO: 1 - SEQ ID NO: 18, and has the advantage of high detection sensitivity for low-concentration pathogens. The present invention uses the SARSeq technology method, combined with the primer combination, to enrich the detection fragments of multiple pathogens through PCR amplification. Compared with the traditional fluorescence quantitative PCR for detecting one or more pathogens, it has the advantages of high detection specificity and non-interference of detection data of each pathogen with each other. The primer combination and its application provided by the present invention have the advantage of low single-sample detection cost. The multiplex primer combination diagnostic technology using the SARSeq technology does not require further construction of a sequencing library and has a lower requirement for subsequent sequencing data volume, making the single-sample detection cost advantage obvious. Detailed implementation manners

[0064] The following combines specific embodiments to further elaborate on the present invention. The following embodiments are not used to limit the present invention, but only to illustrate the present invention. The experimental methods used in the following embodiments, unless otherwise specified, and the experimental methods without specific conditions noted in the embodiments are usually carried out under conventional conditions. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can all be obtained from commercial channels.

[0065] Details of the reagents used in this application are shown in the following table:

[0066]

[0067]

[0068] Example 1

[0069] 1 Reverse transcription

[0070] 1.1 Extraction of sample RNA

[0071] The samples that can be selected in this embodiment are respiratory oropharyngeal and nasopharyngeal swabs under clinical standards and are placed in 3 mL of physiological saline. At the same time, this method is also applicable to the collected patient mouthwash (using 5 mL of physiological saline for gargling).

[0072] The extraction of RNA is completed using a kit, and this kit is a nucleic acid rapid extraction kit. The above samples are mixed with the rapid extraction reagent at a ratio of 1:1 and incubated at 95°C for 5 minutes, and the samples are reserved for use.

[0073] 1.2 Reverse transcription

[0074] Using the extracted RNA as a template, perform a reverse transcription reaction.

[0075] 10×PCR Top buffer A consists of 200 mM Tris-HCL pH8.3, 500 mM KCL, 50 mM MgCl 2 , 200 mM (NH 4 ) 2 SO 4 and 1% Triton X-100.

[0076] The reverse transcription reaction system (25 μL) is shown in the following table:

[0077]

[0078]

[0079] Add the above reverse transcription reaction solution to a 96-well plate, seal it with sealing foil or sealing tape, and perform the reverse transcription reaction. The reaction conditions are set as shown in the following table:

[0080] Temperature Time Number of Cycles 25℃ 5 minutes 1 37℃ 45 minutes 1 85℃ 5 seconds 1 12℃ /

[0081] The extracted RNA undergoes the above reverse transcription reaction to obtain a reverse transcription product.

[0082] 2 First-round PCR reaction

[0083] 2.1 First-round PCR reaction

[0084] Perform the first-round PCR reaction using the reverse transcription product in 1.2 as a template.

[0085] 10×PCR Top buffer D consists of 750 mM Tris-HCL pH8.3, 200 mM (NH 4 ) 2 SO 4 and 1% Triton X-100.

[0086] Mix individual primer pairs to form a primer combination, and the concentration of each primer in the primer combination is 2.5 μM.

[0087] The first-round PCR reaction system (50 μL) is shown in the following table:

[0088]

[0089]

[0090] The first-round PCR reaction conditions are set as shown in the following table:

[0091]

[0092] Taking each 96-well plate as a whole, collect 5 μL of the first-round PCR product mixture from each well (final volume: 96 wells × 5 μL / well), mix well and centrifuge.

[0093] 2.2 Biological purification of PCR products

[0094] Add 2 μL of illustra ExoProStar 1-step to the 5 μL of PCR mixture from the previous step, mix well and centrifuge. The purification conditions are set as shown in the following table:

[0095] Temperature Time Number of Cycles 37℃ 30 minutes 1 80℃ 15 minutes 1 12℃ / /

[0096] After the above steps, the first-round purified PCR products are obtained.

[0097] 3 Second-round PCR reaction

[0098] 3.1 Second-round PCR reaction

[0099] Use the first-round purified PCR products as the template for the second-round PCR, and the primers are PCR2 primers.

[0100] 10×PCR Top buffer E consists of 750 mM Tris-HCL pH 8.3, 200 mM (NH 4 ) 2 SO 4 , 20 mM MgCl 2 and 0.1% Tween-20.

[0101] The second-round PCR reaction system (50 μL) is as shown in the following table:

[0102]

[0103]

[0104] The second-round PCR reaction conditions are set as shown in the following table:

[0105]

[0106] 3.2 Purification of the second-round PCR products

[0107] Collect the second-round PCR products and mix 5 μL / well. Take 20 μL of the final mixture for the next step. Operate according to the Gel Extraction kit Vazyme DC301-1 instruction manual, and collect the DNA products after purification.

[0108] 3.3 Next-generation sequencing of DNA products

[0109] The above-mentioned collected DNA products were sent for next-generation sequencing.

[0110] The primer quality was detected using the analysis software "Mixed Multi-Primer Quality Detection Software" registered by Anku Biotechnology (Suzhou) Co., Ltd., abbreviated as MMPQD (registration number: 2022SR1367530). The non-specific amplification was monitored and analyzed using the "Analysis Software for Non-Specific Amplification Caused by Mixed Multi-Primers", abbreviated as MMNAA (registration number: 2022SR1411973). The multi-pathogen data was analyzed using the "Multi-Pathogen Diagnostic Data Analysis Software Based on General Templates", abbreviated as MDDAS-GT (registration number: 2022SR1562264) and the "Multi-Pathogen Diagnostic Data Analysis Software Based on Degenerate Templates", abbreviated as MDDAS-DT (registration number: 2022SR1560761) to obtain the final results.

[0111] Example 2

[0112] Referring to the experimental method of Example 1, the pathogen detection sensitivity of this method was evaluated. In the experiment, pathogen RNA / DNA standard products with 400, 200, 100, 50, 25, and 12.5 copy numbers were used for evaluation respectively. Each copy number of each standard product included 8 repeated detections. Positive detection rate = number of positive wells in the detection result / 8. The relevant results are shown in the following table:

[0113]

[0114] The results showed that this method could achieve 100% detection in the detection of standard samples of respiratory syncytial virus, influenza B, influenza A, novel coronavirus, Mycoplasma pneumoniae, adenovirus, or rhinovirus at 25, 25, 200, 25, 25, 12.5, and 200 pathogen copy numbers respectively. The relevant data indicated that this method and the primer combination could achieve a very high pathogen detection sensitivity.

Claims

1. A primer combination for simultaneously detecting multiple respiratory pathogens, characterized in that, it includes specific primer sequences; the specific primer sequences consist of SEQ ID NO: 1 - SEQ ID NO:

18.

2. The primer combination according to claim 1, characterized in that, the primer combination further includes an adapter sequence and an Index.

3. The primer combination according to claim 2, characterized in that, the adapter sequence is SEQ ID NO: 19 or SEQ ID NO:

20.

4. The primer combination according to claim 2, characterized in that, 1 - 4 random bases are added upstream of the Index, and the random bases are represented by N.

5. The primer combination according to claim 4, characterized in that, The forward sequences of the said Index are selected from: NCCAATACT, NNTCGCGCAT, NNNGTGTGAAC, NNNNGGTGGCAC, NTAGGTGCT, NNGAGCGGTT, NNNAGGATTAG, NNNNGTGAGCCA, NAATTATGC, NNTTACGCCG, NNNGTAGTGAT, NNNNTGGTTGCA, NGTCCGACC, NNGCCAGCCG, NNNCTCGTGTC, NNNNGCCGTGGC, NGTAGCCAC, NNGACTTATA, NNNGAACGTCG, NNNNATGCGTAG, NAATTAGAT, NNTTACGATA, NNNCTGTACAA, NNNNCGTGGTTG, NAATAACGT, NNTTCTTGAA, NNNGGCAGATC, NNNNCTATGTTA, NGTTGACGC, NNATCTACGA, NNNCTCGACAG, NNNNGAGGCTGC, NCCTCGTAG, NNCATAGGCA, NNNAGATGAAC, NNNNCCGAGTAT, NAATATTGA, NNGTATACCG, NNNGATCCAAC, NNNNAGATACGC, NGGTATCTT, NNCCTCTGGC, NCCATTGTG, NNACTACGGT, NNNAAGTGCTA, NNNNGCCGAACG, NTGTCCACG, NNGACACACT, NNNAATATGCT, NNNNTTCTCATA, NTCTGTGAT, NNCCGAACTT, NNNGTCTAACA, NNNNGACGCCAT, NGCCAATGT, NNCCAACGTC, NNNGTAGATAA, NNNNCTTACGGC, NCCAAGTGC, NNCTAACTCA, NNNAATATCTG, NNNNTTATATCA, NCTGCGGAT, NNGCGGCTTG, NNNGAGTTGAT, NNNNGCACTGAG, NGACCACCT, NAATAGCAA, NCCTACCGG, NNGGAGGATG, NNNCGCTGAAT, NNNNTGTGACGA, NAATAGATT, NNTTAGCGCA, NNNGCGGCCGT, NNNNCAGTAACC, NGCCTAGTA, NNCACGGCGC, NNNGGTGCAGA, NNNNTCGCTGAC, NCAGCCAGT, NNCGTCAACC, NGCCGGCGA, NNGCCTCCGG, NNNAATAGTCC, NNNNTTAGACGT,NGTGGACTA, NNCACGGACG, NNNCACTAGAG, NNNNGCAGATG, NCTCTCACG, NNGGAATCAC, NNNCGTTGACG, NNNNCATCAGGT, NCGTTGTAA, NNGGCACGGT; The reverse sequences of the Index are selected from: AGCTTGGT, AGCTTATG, ATAACGAC, GATGGAGT, TGATCTAA, AGCTATAT, AGCAGAGA, GACGTAAG, AGGCCTGA, TAAGATTA, AGGCGAAT, TTGTTCTT, TACTCCTG, GACTGGCG, ATATATAC, TAGATCGG, AGGCAAGC, TCCAGGTA, AGGTCAAG, TACACTGG, TCCTTGCG, AGGACCTC, TAAGCATT, AGGATATT, AATCGTTA, GTCTACAT, CGCTGCTC, GATCAACA, CGAAGGAC, GATGCCGG, CTACGAAG, GATGCGTC, CTACGGCA, GATTCCTT, CTACTCGA, GATTCGAG, AATCGGCG, TTCGCCGA, CTGGCCTC, GAACTTAT, CGTATTGG, GAAGCACA, CTTAATAC, GAAGTCTT, GAAGAGGC, CGGATAAC, GAATCTGG, CTGATTGA, AATCCGTT, TGCGTACA, GAATCAAT, TGAGTCAG, GAATGCTC, GAATATCC, CTTATGAA, TCGGCACC, AAGAAGCG, CTACGAT, TCGGTCGA, TCGGTAAG, AAGATACA, GTCGCTGT, TCGGATGT, CGAGCCGG, CGATTATC, TCGAAGCT, CTATCATT, AAGTTATC, CGAACGGA, CTACTGAC, TCTTAAGT, TTAGAGTC, AAGACGAA, TTATTATG, CGCTATTA, TCTATCAG, CGGTGGTA, TCACCAAT, CTGGAAGC, CGTAAGAG, AAGAGAGC, TCAACGAG, TGCGAGAC, CCTGGTGT, AAGTAAGT, TGACTGAA, AAGACTGT, CAATGATG, CACAGTAA, TGGTCATT, CAACCGTG, TGGTGCAC, CCACAATG, TGTGTGCC, CACCACGG, TGTGTTAA., 6. A kit for simultaneously detecting multiple respiratory pathogens, characterized in that, the kit includes the primer combination according to any one of claims 1 - 5; the respiratory pathogens are one or more of influenza A virus, influenza B virus, respiratory syncytial virus, adenovirus, novel coronavirus, rhinovirus, and Mycoplasma pneumoniae.

7. The kit according to claim 6, characterized in that, the kit further includes a sample extraction solution, a buffer solution, an enzyme, and dNTPs.

8. The kit according to claim 6, characterized in that, the final concentration of each primer in the primer combination is 0.2 - 1 μM.

9. The kit according to claim 8, characterized in that, the final concentration of each primer in the primer combination is 0.25 μM.

10. The application of the primer combination according to any one of claims 1 - 5 or the kit according to any one of claims 6 - 9 in food detection.

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