Multiple nucleic acid detection primer probe combination and application thereof

Abstract

The invention relates to the technical field of molecular diagnosis, in particular to a multiplex nucleic acid detection primer probe combination and application thereof. A corresponding primer probe combination is designed for researching 10 common respiratory tract pathogens such as new coronavirus, influenza A virus, influenza B virus, adenovirus, rhinovirus, respiratory syncytial virus and human parainfluenza virus types 1, 2, 3 and 4, a respiratory tract infection multiple pathogen detection system is established, a multiple RT-qPCR system is optimized, and the kit is used for detecting the respiratory tract infection multiple pathogens. The optimized system is applied to a clinical throat swab sample for verification, and the multiple pathogen nucleic acid detection kit is researched and developed and used for detecting viral respiratory tract infection. According to the invention, multiple pathogens are detected by adopting a multiple RT-qPCR method, so that the detection time can be shortened, the detection sensitivity and specificity are improved, the detection cost is reduced, and the kit has important clinical significance on rapid diagnosis of respiratory tract multiple pathogen infection.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular diagnosis, and in particular to a multiple nucleic acid detection primer probe combination and application thereof. Background Art

[0002] Most of the test kits for the detection of the new coronavirus on the market are single tests, and there are relatively few nucleic acid test kits for the new coronavirus combined with other common respiratory viruses. The current multiple nucleic acid test kits for the new coronavirus combined with other respiratory viruses mainly detect pathogens including the new coronavirus, influenza A virus, and influenza B virus. However, most of the respiratory virus test kits on the market are for single or several pathogens. If you want to detect multiple pathogens, you need to use multiple sets of different test kits for joint detection, which is inconvenient to operate and costly. In addition, there are relatively few multiple nucleic acid test kits for the new coronavirus combined with other respiratory viruses on the market. The existing multiple test kits can detect fewer types of pathogens, mainly around the new coronavirus, influenza A virus, and influenza B virus. Some researchers have adopted chip technology methods, with the help of rapid detection systems, to detect multiple respiratory viruses at the same time, but the cost is too high and not suitable for clinical application. Therefore, it is urgent to develop a test kit that can detect multiple pathogens at one time. Summary of the invention

[0003] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a multiple nucleic acid detection primer probe combination that can quickly detect 10 common respiratory viruses such as new coronavirus and influenza virus with high sensitivity and specificity.

[0004] To achieve the above object, the technical solution adopted by the present invention is:

[0005] In the first aspect, the present invention provides a multiple nucleic acid detection primer probe combination, comprising primer probe pairs for at least three of the following target genes: SARS-CoV-2RdRp gene, SARS-CoV-2E gene, influenza A virus NS1 gene, influenza B virus HA gene, adenovirus Hexon gene, rhinovirus 5'-UTR untranslated region, respiratory syncytial virus matrix gene, human parainfluenza virus type 1 HN gene, human parainfluenza virus type 2 HN gene, human parainfluenza virus type 3 HN gene, and human parainfluenza virus type 4 Fusion protein.

[0006] The present invention studies 10 common respiratory pathogens, including the new coronavirus, influenza A virus, influenza B virus, adenovirus, rhinovirus, respiratory syncytial virus, human parainfluenza virus types 1, 2, 3, and 4, selects appropriate pathogen target genes, and designs corresponding primer-probe combinations, which can quickly detect the new coronavirus and respiratory infections with high sensitivity and specificity.

[0007] Preferably, the primer probe pair sequence of the SARS-CoV-2RdRp gene is shown in SEQ ID NOs.1-3; the primer probe pair sequence of the SARS-CoV-2E gene is shown in SEQ ID NOs.4-6; the primer probe pair sequence of the influenza A virus NS1 gene is shown in SEQ ID NOs.7-9; the primer probe pair sequence of the influenza B virus HA gene is shown in SEQ ID NOs.10-12; the primer probe pair sequence of the adenovirus Hexon gene is shown in SEQ ID NOs.13-15; the primer probe pair sequence of the rhinovirus 5'-UTR untranslated region is shown in SEQ ID NOs.16-18; the primer probe pair sequence of the respiratory syncytial virus matrix gene is shown in SEQ ID NOs.19-21; the primer probe pair sequence of the human parainfluenza virus type 1 HN gene is shown in SEQ ID NOs.22-24; the primer probe pair sequence of the human parainfluenza virus type 2 HN gene is shown in SEQ ID NOs. NO.25-27; the primer-probe pair sequence of the human parainfluenza virus type 3 HN gene is shown in SEQ ID NO.28-30; the primer-probe pair sequence of the human parainfluenza virus type 4 Fusion protein is shown in SEQ ID NO.31-33.

[0008] When the designed primer-probe combination was subjected to single-plex RT-qPCR reaction, the present invention found that not all primers designed by software can achieve good detection results. In actual detection applications, it is necessary to repeatedly explore the optimal combination of primers and probes to have good specificity and sensitivity. In order to achieve multiplex detection, the present invention compares the primers and probes of each pathogen target gene in pairs, analyzes the interference between primers, hairpin structure and primer dimer, and finally obtains the above-mentioned primer-probe combination. The primer-probe combination designed by the present invention adopts multiplex PCR detection, which can detect at least three pathogens at the same time, has good specificity and a detection sensitivity of 10 1 -10 2 copies / μL.

[0009] Preferably, the probes of the SARS-CoV-2RdRp gene and the SARS-CoV-2E gene are fluorescently labeled with FAM, and the quenching group is BHQ1; the probes of the influenza A virus NS1 gene and the human parainfluenza virus type 2 HN gene are fluorescently labeled with CY5.5, and the quenching group is BHQ3; the probes of the influenza B virus HA gene, the respiratory syncytial virus matrix gene, and the human parainfluenza virus type 3 HN gene are fluorescently labeled with CY5, and the quenching group is BHQ3; the probes of the adenovirus Hexon gene, the rhinovirus 5'-UTR untranslated region, and the human parainfluenza virus type 4 Fusion protein are fluorescently labeled with HEX, and the quenching group is BHQ1; the probe of the human parainfluenza virus type 1 HN gene is fluorescently labeled with FAM, and the quenching group is MGB.

[0010] Preferably, the primer-probe combination further comprises a primer-probe pair of an internal reference gene; the internal reference gene is human epithelial cell ribonuclease RNP; the primer-probe pair sequence of the human epithelial cell ribonuclease RNP is shown in SEQ ID NO.34-36.

[0011] Preferably, the probe of human epithelial cell ribonuclease RNP is fluorescently labeled with CY5.5, and the quenching group is BHQ2.

[0012] In a second aspect, the present invention provides the use of the above-mentioned multiple nucleic acid detection primer probe combination in the preparation of products for detecting new coronavirus infection and / or respiratory pathogen infection.

[0013] In a third aspect, the present invention provides a kit for detecting novel coronavirus infection and / or respiratory pathogen infection, comprising the above-mentioned multiple nucleic acid detection primer probe combination.

[0014] Preferably, the kit further comprises reagents required for multiplex RT-qPCR; the reagents include RT-PCR Buffer, RT-PCR Enzyme Mix, and nuclease-free water.

[0015] Preferably, in the kit, the primer-probe pair comprises a forward primer, a reverse primer and a probe; the final concentration ratio of the forward primer, reverse primer and probe is forward primer: reverse primer: probe = (240-360): (240-360): (72-100).

[0016] More preferably, in the kit, the primer-probe pair comprises a forward primer, a reverse primer and a probe; the final concentration ratio of the forward primer, reverse primer and probe is forward primer: reverse primer: probe = 18:18:5.

[0017] Preferably, the samples detected by the kit include throat swabs and nasal swabs.

[0018] The beneficial effects of the present invention are:

[0019] The present invention studies 10 common respiratory pathogens, including the new coronavirus, influenza A virus, influenza B virus, adenovirus, rhinovirus, respiratory syncytial virus, human parainfluenza virus types 1, 2, 3, and 4, designs corresponding primer and probe combinations, establishes a multiple pathogen detection system for respiratory tract infection, optimizes the multiple RT-qPCR system, and applies the optimized system to clinical pharyngeal swab samples for verification. A multiple pathogen nucleic acid detection kit with high sensitivity and specificity is developed, which can quickly detect 10 common respiratory viruses, including the new coronavirus and influenza virus, and is used to detect viral respiratory infections.

[0020] The kit also contains matching reagent components for use in multiple RT-qPCR experiments, such as 2×RT-PCR Buffer, 25×RT-PCR Enzyme Mix, and nuclease-free water (from the one-step RT-qPCR kit AgPath-ID TM The reagents in the One-Step RT-PCR Kit (#4387391) were used. After the sample was collected, nucleic acid was extracted. The extracted nucleic acid was used as a template. Mix solutions of each group except nucleic acid were prepared according to the reaction system. Then, the mixed solutions were added to the qPCR 96-well plate (Bio-Rad, #HSP9601). Then, the template nucleic acid was added. The reaction program was set using the fluorescent quantitative PCR instrument Bio-Rad CFX96 for reaction. The reaction time was about 1 hour and 40 minutes.

[0021] The multiple pathogen nucleic acid detection kit of the present invention adopts the multiple RT-qPCR method to detect multiple pathogens, which can shorten the time required for detection, improve detection sensitivity and specificity, and reduce detection costs. It has important clinical significance for the rapid diagnosis of multiple pathogen infections in the respiratory tract. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The single-plex RT-qPCR results for the primer-probe combinations in Table 1 are shown.

[0023] Figure 2 Single-plex RT-qPCR results when the quencher group of the human parainfluenza virus type 1 HN gene probe is BHQ1.

[0024] Figure 3 Single-plex RT-qPCR results for SARS-CoV-2, IAV, and IBV.

[0025] Figure 4These are the single-plex RT-qPCR results for ADV, HRV, RSV, and HPIV1.

[0026] Figure 5 These are the single-plex RT-qPCR results for HPIV2, HPIV3, and HPIV4.

[0027] Figure 6 The multiplex RT-qPCR results of group 1 when the final concentration (nM) of forward primer: reverse primer: probe was 360:360:100.

[0028] Figure 7 The multiplex RT-qPCR results of group 2 when the final concentration (nM) of forward primer: reverse primer: probe was 360:360:100.

[0029] Figure 8 The multiplex RT-qPCR results of group 3 when the final concentration (nM) of forward primer: reverse primer: probe was 360:360:100.

[0030] Fig. 9 The multiplex RT-qPCR results of each group when the final concentration (nM) of forward primer: reverse primer: probe was 320:320:100.

[0031] Fig.10 The multiplex RT-qPCR results of each group when the final concentration (nM) of forward primer: reverse primer: probe was 280:280:88.

[0032] Fig.11 The multiplex RT-qPCR results of each group when the final concentration (nM) of forward primer: reverse primer: probe was 240:250:72.

[0033] Fig.12 The multiplex RT-qPCR results of each group when the annealing extension temperature was 50°C.

[0034] Fig.13 The multiplex RT-qPCR results of each group when the annealing extension temperature was 55°C.

[0035] Fig.14 The multiplex RT-qPCR results of each group when the annealing extension temperature was 65°C.

[0036] Fig.15 The multiplex RT-qPCR results of each group when the annealing extension temperature was 56°C.

[0037] Fig.16 The multiplex RT-qPCR results of each group when the annealing extension temperature was 58°C.

[0038] Fig.17 This is the multiplex RT-qPCR result of group 2 in the multiplex RT-qPCR grouping in Table 10.

[0039] Fig.18 This is the multiplex RT-qPCR result of group 3 in the multiplex RT-qPCR grouping in Table 10.

[0040] Fig.19 It is the specific detection result of cross-reaction between groups.

[0041] Fig. 20 It is the specific detection result of cross-reaction within the group. DETAILED DESCRIPTION

[0042] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0043] Example 1: Design of primer-probe pairs

[0044] The present invention studies 10 common respiratory pathogens, including novel coronavirus, influenza A virus, influenza B virus, adenovirus, rhinovirus, respiratory syncytial virus, human parainfluenza virus types 1, 2, 3, and 4, selects target genes of pathogens, searches for sequences of target genes corresponding to each of the above pathogens based on reading relevant literature, designs corresponding primer-probe combinations including three sequences of forward primer, reverse primer and probe, and uses software such as Oligo 7 and Primer Premier to analyze interference between primers, hairpin structure and primer dimer.

[0045] When performing single-plex RT-qPCR reactions on the designed primer-probe combinations, the present invention found that not all primers designed by software can achieve good detection results. In actual detection applications, it is necessary to repeatedly explore the optimal combination of primers and probes to have good specificity and sensitivity.

[0046] 1. In the early stage of the experiment, the present invention designed the pathogen target genes and their corresponding primer probe combinations as shown in Table 1:

[0047] Table 1 Primer and probe sequences of target genes of various pathogens (F: forward primer; R: reverse primer)

[0048]

[0049]

[0050] The gene sequence between the two target sites of the pathogen forward and reverse primers was used as the detection target gene sequence, and then the corresponding plasmid was artificially synthesized in vitro. All primers, probes, and plasmids were synthesized by Sangon Biotech (Shanghai) Co., Ltd. The probe purification method was HPLC, and the primer purification method was PAGE. 2. Single-plex RT-qPCR reaction sensitivity test was performed on the primer-probe combination in Table 1:

[0051] Use a plasmid extraction kit to extract plasmid DNA, perform 1.0% agarose gel electrophoresis analysis on plasmid DNA, check the fragment size, send the positive plasmid that matches the expected fragment size to the relevant company for sequencing, perform BLAST comparison on the NCBI GenBank database, search for homologous nucleic acid sequences, and determine the species to which the nucleic acid belongs. The identified plasmid is stored in glycerol and stored at -80°C for future use.

[0052] For the positive plasmid DNA that was successfully sequenced, the DNA concentration was measured using an ultraviolet spectrophotometer, and its original copy number was calculated according to the following formula:

[0053]

[0054] Among them, ds DNA MV=nucleotides×660 Dolton / bp; ss DNA MV=nucleotides×330 Dolton / bp; ss RNA MV=nucleotides×340 Dolton / bp.

[0055] Then, enzyme-free water was used to dilute the mixture 10-fold to different concentrations (10 8 ~10 0 copies / μL) as a series of standards for nucleic acid detection of various pathogens and stored at -80°C. Using the plasmid DNA standard containing cloned genes of pathogens as templates, according to AgPath-ID TM One-Step RT-PCR Kit (Applied Biosystems #4387391) Kit instructions for setting up a single-plex real-time fluorescence quantitative RT-PCR (RT-qPCR) reaction system are shown in Table 2, and the reaction conditions are shown in Table 3.

[0056] Table 2 RT-qPCR reaction system

[0057]

[0058] Table 3 RT-qPCR reaction conditions

[0059]

[0060] The results of single-plex PCR of the primer-probe pairs designed in Table 1 are as follows Figure 1 The results showed that the sensitivity of different primer-probe combinations varied greatly. The sensitivity of IAV was as high as 10 4 , the highest sensitivity of IBV is 10 2 , ADV's maximum sensitivity is 10 3 The sensitivity of HPIV2 is up to 10 3 The amplification curve of HPIV3 is rather chaotic and the results are not very credible.

[0061] In addition, the present invention also found that the quenching group of the human parainfluenza virus type 1 HN gene probe also affects the detection effect. When the quenching group of the pathogen probe is BHQ1, no CT value is measured during the detection process ( Figure 2 ), the CT value can only be detected when it is adjusted to the MGB group.

[0062] 3. The present invention continuously tests and screens out primer-probe combinations with better detection effects in combination with single-plex PCR detection. For pathogens with low sensitivity, the primers, probe sequences and target gene sequences are re-searched to improve the detection sensitivity and continuously optimized. Finally, the RdRp gene and E gene of the new coronavirus, the NS1 non-structural protein of influenza A virus, the HA hemagglutinin gene of influenza B virus, the Hexon hexon gene of adenovirus, the 5'-UTR untranslated region of rhinovirus, the HA hemagglutinin gene of human parainfluenza virus type 1, 2, and 3, and the fusion glycoprotein Fusion protein of human parainfluenza virus type 4 are selected as target genes. Finally, the primer-probe pairs shown in Table 4 are selected. The target gene sequences of each pathogen are shown in Table 5.

[0063] Test results such as Figure 3-5 As shown, the sensitivity of the 10 pathogens reached 10 1 copies / μL.

[0064] Table 4 Primer and probe sequences of target genes of various pathogens (F: forward primer; R: reverse primer)

[0065]

[0066]

[0067]

[0068] Table 5 Target gene sequences of various pathogens

[0069]

[0070]

[0071] Example 2: Exploration of multiplex RT-qPCR detection system

[0072] In this example, a multiplex RT-qPCR detection system was established based on the single-plex RT-qPCR detection system.

[0073] In order to improve product specificity and reduce nonspecific competitive inhibition between primers and probes in the reaction components, the multiplex RT-qPCR reaction system and conditions of this embodiment are optimized, including exploring the annealing extension temperature, screening the optimal concentration ratio of primers and probes, adjusting the template concentration, changing the number of cycles, etc. The obtained amplified product is subjected to 3.0% agarose gel electrophoresis to detect the product size and compared with the expected fragment size. Then the multiplex RT-qPCR is tested for sensitivity, specificity, repeatability and consistency. An optimized reaction system is obtained.

[0074] 1. In order to facilitate rapid detection, the present invention sets up a three-tube four-fold group detection, and the nucleic acid of the same sample is added to three detection tubes (i.e., 3 groups), and each tube detects 3-4 pathogens at the same time (any group detects the internal reference gene, except that the group where the internal reference gene is located detects 3 pathogens, and the other two groups detect 4 pathogens). The target gene probes of the 4 pathogens detected in each tube need to be labeled with different fluorescent groups. The present invention sets the grouping shown in Table 6, and the multiplex RT-qPCR detection reaction system and reaction conditions shown in Tables 7 and 8.

[0075] Table 6 Multiple RT-qPCR grouping

[0076]

[0077] Table 7 Multiplex RT-qPCR (quadruple) reaction system

[0078]

[0079]

[0080] Note: The initial concentration of the forward / reverse primers is 20 μM, and the initial concentration of the probe is 10 μM. After calculation, in system 1, the final concentration (nM) of the forward primer: reverse primer: probe is 360:360:100; in system 2, the final concentration (nM) of the forward primer: reverse primer: probe is 320:320:100;

[0081] In system 3, the final concentrations (nM) of forward primer:reverse primer:probe were 280:280:88;

[0082] In system 4, the final concentrations (nM) of forward primer: reverse primer: probe are 240:250:72.

[0083] Table 8 Multiplex RT-qPCR reaction conditions

[0084]

[0085] The groups in Table 6 were amplified, and the amplification effects of different systems were as follows: Figure 6-11 shown.

[0086] Table 9

[0087]

[0088] The results of the final concentration ratios of primers and probes with different ratios are shown in Table 9. The results showed that when the ratio of primers and probes was 360:360:100 (i.e., reaction system 1), the amplification effect was the best ( Figure 6-8 ).

[0089] 2. Exploration of annealing extension temperature:

[0090] Based on Table 8, the appropriate annealing extension temperature was explored. Five annealing extension temperatures were set for amplification at 50°C, 55°C, 65°C, 56°C, and 58°C. The amplification effects of different annealing extension temperatures are shown in the figure. Figure 12-16 shown.

[0091] The results showed that when the annealing extension temperature was 60°C (Table 8), the amplification effect was the best. Therefore, the reaction conditions in Table 8 were used for subsequent research.

[0092] 3. In addition to the grouping in Table 6, the present invention also designs the grouping in Table 10 for detection.

[0093] Table 10 Multiple RT-qPCR grouping

[0094]

[0095]

[0096] Multiple RT-qPCR sensitivity tests were performed according to the groups of 6 and 10. The plasmid DNA standards of each pathogen in each group were used as templates, and the concentration gradient was set to 10 8 copies / μL up to 10 0The samples were amplified on a Bio-Rad CFX96 fluorescence quantitative PCR instrument. The logarithm of the standard concentration (copies / μL) was used as the horizontal axis and the threshold cycle number (Ct, Cycle Threshold) was used as the vertical axis to draw a standard curve, and the amplification efficiency was calculated; the amplification curve was drawn with the relative fluorescence units (RFU, Relative fluorescence units) as the vertical axis and the corresponding cycle number as the horizontal axis.

[0097] Table 6 Grouped test results ( Figure 6-8 ) showed that among the first group of pathogens, the sensitivity of the RdRp gene of SARS-CoV-2 was 10 2 Except for 10 copies / μL, the sensitivity of the other three pathogens IAV, IBV, and HRV was 10 1 In the second group of pathogens, the minimum detection limit of SARS-CoV-2 E gene, ADV, and HPIV3 was 10 2 Among the third group of pathogens, the minimum detection limit of HPIV1, HPIV2, HPIV4, and RSV was 10 1 copies / μL.

[0098] The test results of the groups in Table 10 are as follows Figure 17-18 As shown, the sensitivity of the first group of pathogens is 10 1 (The results are the same Figure 6 ); the sensitivity of the second group of pathogens is 10 3 ( Fig.17 ); in the detection of pathogens in the third group, the sensitivity of HPIV1 and HPIV2 was 10 3 The sensitivity of HPIV3 is 10 2 The sensitivity of HPIV4 is 10 1 ( Fig.18 ). Therefore, the grouping detection sensitivity of Table 6 is better.

[0099] In summary, the detection system for the best multiplex RT-qPCR test obtained in this embodiment is: the detection grouping is shown in Table 6, the reaction system is system 1, and the reaction conditions are shown in Table 8. The present invention subsequently uses this detection system for experiments.

[0100] Example 4: Multiplex RT-qPCR Assay

[0101] The optimal reaction system obtained in Example 3 was tested for specificity, repeatability and consistency.

[0102] 1. Multiplex RT-qPCR specificity test

[0103] To each reaction group, a concentration of 10 4 The plasmid DNA standard mixture of several respiratory pathogens other than this group of pathogens at 100 copies / μL was used as a template to check whether there was any cross-reaction between groups. The cross-reaction within the group was tested by the single-plex RT-qPCR reaction system of the corresponding pathogens.

[0104] The results are as follows Figure 19-20 As shown, the remaining pathogens and negative controls (nuclease-free water) had no positive amplification signals, indicating that each reaction group had no cross-reaction with other detected pathogens, and the specificity of the multiplex RT-qPCR test method was good.

[0105] 2. Multiplex RT-qPCR repeatability test

[0106] Repeatability test was performed according to the grouping in Table 6: plasmid DNA standard of each pathogen in each group was used as template to dilute to different concentrations, and the concentration gradient was set to 10 7 , 10 5 and 10 3 The multiple detection method established above was used to repeat the detection three times in parallel, and the coefficient of variation CV (%) was obtained by analysis. The results are shown in Table 11.

[0107] Table 11 Multiple RT-qPCR repeatability test results

[0108]

[0109] The results showed that the coefficient of variation of the Ct values ​​of each group was 0.24% to 2.24%, all less than 5%, indicating that the detection method had good repeatability.

[0110] 3. Multiple RT-qPCR consistency test

[0111] Multiplex RT-qPCR was used to detect clinical samples with known pathogen diagnosis results, and the test results were compared and analyzed using the Kappa test to evaluate the clinical detection effect of this detection method.

[0112] If Kappa ≥ 0.75, it means that the diagnostic results of the two methods are consistent; if 0.4 ≤ Kappa < 0.75, it means that the diagnostic results of the two methods are generally consistent; if Kappa < 0.4, it means that the diagnostic results of the two methods are inconsistent. The results are shown in Table 12.

[0113] Table 12 Comparison of pathogen detection results of clinical samples

[0114]

[0115] The results showed that the Kappa values ​​corresponding to each pathogen were ≥0.75, indicating that the multiplex RT-qPCR method provided by the present invention had good consistency with the detection results of the disease control department.

[0116] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A multiplex nucleic acid detection primer probe combination, characterized in that: The invention comprises primer-probe pairs for at least three of the following target genes: SARS-CoV-2RdRp gene, SARS-CoV-2E gene, influenza A virus NS1 gene, influenza B virus HA gene, adenovirus Hexon gene, rhinovirus 5'-UTR untranslated region, respiratory syncytial virus matrix gene, human parainfluenza virus type 1 HN gene, human parainfluenza virus type 2 HN gene, human parainfluenza virus type 3 HN gene, and human parainfluenza virus type 4 Fusion protein.

2. The multiple nucleic acid detection primer-probe combination according to claim 1, characterized in that: The primer probe pair sequence of the SARS-CoV-2RdRp gene is shown in SEQ ID NO.1-3; the primer probe pair sequence of the SARS-CoV-2E gene is shown in SEQ ID NO.4-6; the primer probe pair sequence of the influenza A virus NS1 gene is shown in SEQ ID NO.7-9; the primer probe pair sequence of the influenza B virus HA gene is shown in SEQ ID NO.10-12; the primer probe pair sequence of the adenovirus Hexon gene is shown in SEQ ID NO.13-15; the primer probe pair sequence of the rhinovirus 5'-UTR untranslated region is shown in SEQ ID NO.16-18; the primer probe pair sequence of the respiratory syncytial virus matrix gene is shown in SEQ ID NO.19-21; the primer probe pair sequence of the human parainfluenza virus type 1 HN gene is shown in SEQ ID NO.22-24; The primer-probe pair sequences of the human parainfluenza virus type 2 HN gene are shown in SEQ ID NOs.25-27; the primer-probe pair sequences of the human parainfluenza virus type 3 HN gene are shown in SEQ ID NOs.28-30; and the primer-probe pair sequences of the human parainfluenza virus type 4 Fusionprotein are shown in SEQ ID NOs.31-33.

3. The multiple nucleic acid detection primer-probe combination according to claim 1, characterized in that: The probes of the SARS-CoV-2RdRp gene and the SARS-CoV-2E gene are fluorescently labeled with FAM, and the quenching group is BHQ1; the probes of the influenza A virus NS1 gene and the human parainfluenza virus type 2 HN gene are fluorescently labeled with CY5.5, and the quenching group is BHQ3; the probes of the influenza B virus HA gene, the respiratory syncytial virus matrix gene, and the human parainfluenza virus type 3 HN gene are fluorescently labeled with CY5, and the quenching group is BHQ3; the probes of the adenovirus Hexon gene, the rhinovirus 5'-UTR untranslated region, and the human parainfluenza virus type 4 Fusion protein are fluorescently labeled with HEX, and the quenching group is BHQ1; the probe of the human parainfluenza virus type 1 HN gene is fluorescently labeled with FAM, and the quenching group is MGB.

4. The multiple nucleic acid detection primer-probe combination according to claim 1, characterized in that: The primer-probe combination also includes a primer-probe pair of an internal reference gene; the internal reference gene is human epithelial cell ribonuclease RNP; the primer-probe pair sequence of the human epithelial cell ribonuclease RNP is shown in SEQ ID NO.34-36.

5. The multiple nucleic acid detection primer-probe combination according to claim 4, characterized in that: The probe of human epithelial cell ribonuclease RNP is fluorescently labeled with CY5.5, and the quenching group is BHQ2.

6. Use of the multiple nucleic acid detection primer probe combination as described in any one of claims 1-5 in the preparation of products for detecting new coronavirus infection and / or respiratory pathogen infection.

7. A kit for detecting novel coronavirus infection and / or respiratory pathogen infection, characterized in that: It comprises a multiple nucleic acid detection primer-probe combination as described in any one of claims 1-5.

8. The kit according to claim 7, characterized in that The kit also includes reagents required for multiplex RT-qPCR; the reagents include RT-PCR Buffer, RT-PCR Enzyme Mix, and nuclease-free water.

9. The kit according to claim 7, characterized in that In the kit, the primer-probe pair includes a forward primer, a reverse primer and a probe; the final concentration ratio of the forward primer, reverse primer and probe is forward primer: reverse primer: probe = (240-360): (240-360): (72-100).

10. The kit according to claim 7, characterized in that The samples tested by the kit include throat swabs and nasal swabs.

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