A set of molecular targets, primer compositions, methods and uses thereof for the identification of respiratory pathogens

Abstract

The application discloses a set of molecular targets, primer compositions, methods and applications for identifying respiratory pathogens, the respiratory pathogens are novel coronavirus, influenza A virus and respiratory syncytial virus, and nucleotide sequences of the molecular targets are shown as SEQ ID NO:1-3. The application provides molecular targets, primers and probes for detecting three respiratory pathogens, and establishes a triple real-time RT-RAA detection technology, which can realize rapid identification and differentiation of three respiratory infectious disease pathogens with similar clinical symptoms within 30 minutes, reduces the operation threshold of the existing detection technology, realizes the target of rapidly, simply and accurately identifying and differentiating the three viruses, meets the needs of early clinical diagnosis of infection, provides technical support for precise medication, and has wide application value.

Description

Technical Field

[0001] This invention relates to the field of virus detection technology, and more specifically, to a set of molecular targets, primer compositions, methods, and applications for identifying respiratory pathogens. Background Technology

[0002] The novel coronavirus (Severe acute respiratory syndrome coronavirus-2, SARS-CoV-2), influenza A virus (IAV), and respiratory syncytial virus (RSV) are common respiratory pathogens that cause very similar clinical symptoms, including fever, cough, and fatigue, making them difficult to distinguish based on symptoms alone. Rapid identification of these three viruses in the early stages of infection, followed by targeted medication, is crucial for improving clinical survival rates. Therefore, there is an urgent need for a detection technology that can rapidly, specifically, and accurately differentiate between these three viruses to meet the needs of early clinical diagnosis and treatment.

[0003] Reverse transcription polymerase chain reaction (RT-qPCR) is a commonly used technique for diagnosing respiratory pathogens, and existing techniques have established RT-qPCR detection methods for SARS-CoV-2, IAV, and RSV. However, the adaptive evolution of these viruses often leads to mutations that allow them to evade the detection primers and probes designed for current RT-qPCR methods, resulting in false negative results. Furthermore, the implementation of RT-qPCR requires trained technicians and expensive laboratory equipment, which presents a significant challenge in areas with limited medical resources but a higher risk of infectious diseases. Therefore, there is an urgent need to develop a rapid detection technique that is simple to operate and requires no special equipment.

[0004] Reverse transcription-recombinase-aided amplification (RT-RAA) is a novel isothermal in vitro nucleic acid amplification technique. It first uses reverse transcriptase to reverse transcribe RNA samples into cDNA, and then uses recombinase to bind primers and probes to mediate a strand displacement reaction, achieving real-time amplification and detection. Detection can be completed within 30 minutes at 37–42°C. It features high sensitivity, strong specificity, short reaction time, simple operation, intuitive result interpretation, and suitability for rapid on-site diagnosis, and has been applied to the single-target detection of various pathogenic microorganisms. However, there are few reports on multiplex RT-RAA detection technologies that can simultaneously detect SARS-CoV-2, IAV, and RSV. This is mainly because the target sequence of the target gene detected by RAA technology is relatively short, while the genomic sequences of these three pathogens have high similarity. RAA amplification primers designed based on commonly used molecular targets are prone to non-specific amplification, affecting detection accuracy. Furthermore, as the number of detection targets increases, the optimization and balance of multiplex RAA detection systems become more complex, requiring consideration of issues such as primer pair interactions, detection and differentiation of amplification products. Summary of the Invention

[0005] To address the aforementioned problems in the prior art, this invention provides a set of molecular targets, primer compositions, methods, and applications for identifying respiratory pathogens.

[0006] The first objective of this invention is to provide a set of molecular targets for identifying respiratory pathogens.

[0007] A second objective of this invention is to provide the application of reagents for detecting the molecular target in the preparation of products for identifying respiratory pathogens.

[0008] A third objective of this invention is to provide primers for detecting the molecular target.

[0009] A fourth object of the present invention is to provide a primer composition for detecting the molecular target.

[0010] A fifth object of the present invention is to provide a primer-probe composition for detecting the molecular target.

[0011] A sixth object of the present invention is to provide the use of the primers, the primer compositions, or the primer-probe compositions in the preparation of products for identifying respiratory pathogens.

[0012] The seventh objective of this invention is to provide a method for identifying respiratory pathogens for the purpose of non-disease treatment diagnosis.

[0013] The eighth objective of this invention is to provide an RT-RAA detection system for identifying respiratory pathogens.

[0014] To achieve the above objectives, the present invention is implemented through the following solution:

[0015] Based on newly discovered molecular targets, this invention establishes a triple real-time RT-RAA detection method to rapidly and accurately identify three respiratory pathogens with similar clinical symptoms: influenza A virus, novel coronavirus, and respiratory syncytial virus.

[0016] A set of molecular targets for identifying respiratory pathogens, including novel coronavirus, influenza A virus, and respiratory syncytial virus (RSV), with nucleotide sequences shown in SEQ ID NO:1–3, respectively. Specifically, the nucleotide sequence of the target sequence for novel coronavirus is shown in SEQ ID NO:1; the nucleotide sequence of the target sequence for influenza A virus is shown in SEQ ID NO:2; and the nucleotide sequence of the target sequence for RSV is shown in SEQ ID NO:3.

[0017] The application of the reagents for the molecular targets in the preparation of products for identifying respiratory pathogens should also be within the scope of protection of this invention.

[0018] Primers for detecting the molecular target are shown in SEQ ID NO:4–13, SEQ ID NO:15–24, SEQ ID NO:26–35, and SEQ ID NO:37–59. Specifically, primers with nucleotide sequences as shown in SEQ ID NO:4–13 and SEQ ID NO:37–44 detect the novel coronavirus; primers with nucleotide sequences as shown in SEQ ID NO:15–24 and SEQ ID NO:45–51 detect influenza A virus; and primers with nucleotide sequences as shown in SEQ ID NO:26–35 and SEQ ID NO:52–59 detect respiratory syncytial virus.

[0019] A primer composition for detecting the molecular target comprises primer pairs 1 to 6; primer pair 1 is a primer with nucleotide sequences as shown in SEQ ID NO:6 and SEQ ID NO:10; primer pair 2 is a primer with nucleotide sequences as shown in SEQ ID NO:18 and SEQ ID NO:23; primer pair 3 is a primer with nucleotide sequences as shown in SEQ ID NO:27 and SEQ ID NO:32; primer pair 4 is a primer with nucleotide sequences as shown in SEQ ID NO:39 and SEQ ID NO:10; primer pair 5 is a primer with nucleotide sequences as shown in SEQ ID NO:47 and SEQ ID NO:23; and primer pair 6 is a primer with nucleotide sequences as shown in SEQ ID NO:27 and SEQ ID NO:58. Primer pairs 1 and 4 are used to detect novel coronavirus; primer pairs 2 and 5 are used to detect influenza A virus; and primer pairs 3 and 6 are used to detect respiratory syncytial virus.

[0020] A primer-probe composition for detecting the molecular target, comprising primers and a probe, wherein the primers are primers for detecting the molecular target or a primer composition for detecting the molecular target. Preferably, the probe is an exo probe.

[0021] Preferably, the nucleotide sequences of the probes are shown in SEQ ID NO:14, 25, and 36. Specifically, the probe with the nucleotide sequence shown in SEQ ID NO:14 detects the novel coronavirus; the probe with the nucleotide sequence shown in SEQ ID NO:25 detects influenza A virus; and the probe with the nucleotide sequence shown in SEQ ID NO:36 detects respiratory syncytial virus.

[0022] More preferably, the primers are primer sets for detecting the molecular target, and the primer-probe composition comprises primer-probe compositions 1 to 3; primer-probe composition 1 comprises primer pair 1 or primer pair 4, and a probe with a nucleotide sequence as shown in SEQ ID NO: 14, for detecting novel coronavirus; primer-probe composition 2 comprises primer pair 2 or primer pair 5, and a probe with a nucleotide sequence as shown in SEQ ID NO: 25, for detecting influenza A virus; primer-probe composition 3 comprises primer pair 3 or primer pair 6, and a probe with a nucleotide sequence as shown in SEQ ID NO: 36, for detecting respiratory syncytial virus.

[0023] More preferably, the primer-probe composition 1 comprises primer pair 4 and a probe with a nucleotide sequence as shown in SEQ ID NO:14, for detecting novel coronavirus; the primer-probe composition 2 comprises primer pair 5 and a probe with a nucleotide sequence as shown in SEQ ID NO:25, for detecting influenza A virus; and the primer-probe composition 3 comprises primer pair 6 and a probe with a nucleotide sequence as shown in SEQ ID NO:36, for detecting respiratory syncytial virus.

[0024] More preferably, the probes in each of the primer-probe compositions 1 to 3 have different fluorescent groups, and the fluorescent groups are TAMRA, FAM, or ROX.

[0025] The application of the primers, primer compositions, or primer-probe compositions in the preparation of products for identifying respiratory pathogens should also be within the scope of protection of this invention.

[0026] A method for identifying respiratory pathogens for non-disease treatment diagnosis purposes, comprising detection using the primers, the primer composition, or the primer-probe composition, and the RT-RAA method.

[0027] An RT-RAA detection system for identifying respiratory pathogens comprises the primers, the primer composition or the primer-probe composition and RAA reagents.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] This invention provides molecular targets, primers, and probes for detecting three respiratory pathogens, and establishes a triple real-time RT-RAA detection technology that can rapidly distinguish and identify three respiratory infectious disease pathogens with similar clinical symptoms within 30 minutes. This reduces the operational threshold of existing detection technologies, achieves the goal of rapid, simple, and accurate differentiation and identification of three viruses, meets the needs of early clinical diagnosis of infection, provides technical support for precision medicine, and has broad application value. Attached Figure Description

[0030] Figure 1 The positions of the RT-RAA primer and exo probe on the M gene sequence of different IAV strains are shown; the boxed areas represent the corresponding matching regions of the forward primer, exo probe, and reverse primer, respectively; solid and hollow triangles mark two T residues in the fluorescent and quenching groups of the exo probe, respectively; the arrows indicate tetrahydrofuran.

[0031] Figure 2The positions of the RT-RAA primer and exo probe on the N gene sequence of different SARS-CoV-2 strains are shown; the boxes cover the corresponding matching regions of the forward primer, exo probe, and reverse primer, respectively; solid triangles and hollow triangles mark two T residues in the fluorescent and quenching groups of the exo probe, respectively; the arrows mark tetrahydrofuran.

[0032] Figure 3 The positions of the RT-RAA primers and probes on the N gene sequences of different RSV strains are shown; the boxes cover the corresponding matching regions of the forward primer, exo probe, and reverse primer, respectively; solid triangles and hollow triangles mark the two T residues in the fluorescent and quenching groups of the exo probe, respectively; the arrows mark tetrahydrofuran.

[0033] Figure 4 Primers for triple real-time RT-RAA detection of SARS-CoV-2 were determined; A is a schematic diagram of the principle of the first round of primer screening, in which the numbers in the primer names indicate the position in the N gene of SARS-CoV-2 (GenBank accession number: MW001266.1); B is the first round screening result of the reverse primers; C is the first round screening result of the forward primers; D is a schematic diagram of the principle of the second round of primer screening; E is the second round screening result of the reverse primers; F is the second round screening result of the forward primers.

[0034] Figure 5 Primers for IAV triple real-time RT-RAA detection were determined; A is a schematic diagram of the principle of the first round of primer screening, where the numbers in the primer names indicate the position in the M gene of IAV (GenBank accession number: MN570352.1); B shows the first round of screening results for the reverse primers; C shows the first round of screening results for the forward primers; D is a schematic diagram of the principle of the second round of primer screening; E shows the second round of screening results for the reverse primers; and F shows the second round of screening results for the forward primers.

[0035] Figure 6 Primers for RSV triple real-time RT-RAA detection were determined; A is a schematic diagram of the primer principle for the first round of screening, where the numbers in the primer names indicate the position in the RSV N gene (GenBank accession number: MN310477.1); B shows the results of the first round of screening for reverse primers; C shows the results of the first round of screening for forward primers; D is a schematic diagram of the primer principle for the second round of screening; E shows the results of the second round of screening for reverse primers; F shows the results of the second round of screening for forward primers.

[0036] Figure 7The results of the specificity evaluation of the triple real-time RT-RAA method are shown. Curves 1 to 14 correspond to the templates of IAV, SARS-CoV-2, RSV, HPIV1, HPIV3, HMPV, HADV, HRV, HCoV, C. Pneumonia, M. Pneumonia, IBV, HBoV1 and negative control, respectively.

[0037] Figure 8 The results show the sensitivity evaluation of the triple real-time RT-RAA method; curves 1-5 correspond to 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL and 10 0 The standard plasmid dilution solution was 1 copies / μL, and curve 6 corresponds to the negative control; A and B are the results of IAV detection by triple real-time RT-RAA and triple RT-qPCR, respectively; C and D are the results of SARS-CoV-2 detection by triple real-time RT-RAA and triple RT-qPCR, respectively; E and F are the results of RSV detection by triple real-time RT-RAA and triple RT-qPCR, respectively. Detailed Implementation

[0038] The present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods; the materials and reagents used, unless otherwise specified, are commercially available.

[0039] Example 1: Determination of molecular targets, primers, and probes for triple real-time RT-RAA

[0040] 1. Determination of molecular targets

[0041] The DNASTAR software was used to compare and analyze the SARS-CoV-2N gene sequences of 30 different strains, the IAVM gene sequences of 12 different strains, and the RSV N gene sequences of 31 different strains. The results are as follows: Figure 3 As shown, molecular targets for detecting SARS-CoV-2 (nucleotide sequence as shown in SEQ ID NO:1), molecular targets for IAV (nucleotide sequence as shown in SEQ ID NO:2), and molecular targets for RSV (nucleotide sequence as shown in SEQ ID NO:3) were obtained.

[0042] 2. Primer and probe design and determination of optimal primer combinations

[0043] Primers and exo probes were designed for the three obtained molecular targets using SnapGene software (version 3.6.2022), and the results were analyzed based on TwistAmp. TM The amplification guidelines are used to screen for the best primer combinations.

[0044] (1) First round of screening

[0045] Five forward primers and five reverse primers were designed for the exo probe of each molecular target, and the specific sequences are shown in Table 1.

[0046] Table 1. Sequence information of primers and exo probes selected in the first round of screening.

[0047]

[0048]

[0049] FAM-dT: Thymidine nucleotide carrying FAM; TAMRA-dT: Thymidine nucleotide carrying TAMRA; ROX-dT: Thymidine nucleotide carrying ROX; BHQ1-dT: Thymidine nucleotide carrying BHQ1; BHQ2-dT: Thymidine nucleotide carrying BHQ2; THF: Tetrahydrofuran; C3-Spacer: C3-Spacer blocks elongation at the 3' end. Carboxyfluorescein (FAM); Tetramethylrhodamine (TAMRA); Rhodamine X (ROX).

[0050] The screening method is as follows: For each pathogen, one forward primer is randomly selected from five forward primers, and then one reverse primer is randomly selected from five reverse primers for RT-RAA reaction. The RT-RAA method uses a kit (#S002ZC) from Hangzhou Zhongce Biotechnology Co., Ltd. (China). The RT-RAA system (50 μL / reaction) consists of the following components: Buffer A (25 μL); one forward primer (10 μM, 4.0 μL); one reverse primer (10 μM, 4.0 μL); one exo probe (10 μM, 1.8 μL); DEPC water (8.7 μL); template (4.0 μL); Buffer B (2.5 μL). The RT-RAA reaction program is as follows: place the reaction tube in a QuantStudio real-time PCR System (Applied Biosystems, America) and incubate at 42°C for 20 minutes (1 cycle / min), monitoring the fluorescence signal in real time.

[0051] The templates for each pathogen are as follows: The template for SARS-CoV-2 is the standard plasmid of the SARS-CoV-2N gene, denoted as pUC57-SARS-CoV-2-N, which is obtained by ligating the N gene sequence of SARS-CoV-2 (MW001266.1) into the pUC57 vector; the template for IAV is the standard plasmid of the IAV M gene, denoted as pUC57-IAV-M, which is obtained by ligating the M gene sequence of IAV (MN570352.1) into the pUC57 vector; the template for RSV is the standard plasmid of the RSV N gene, denoted as pUC57-RSV-N, which is obtained by ligating the N gene sequence of RSV (MN310477.1) into the pUC57 vector.

[0052] For SARS-CoV-2, such as Figure 4 As shown in A to C, the optimal reverse primer was determined to be R940-969 (SEQ ID NO:10) using the forward primer F768-797 (SEQ ID NO:5). Then, the optimal primer combination was determined to be the forward primer F804-833 (SEQ ID NO:6) and the reverse primer R940-969 (SEQ ID NO:10) using the reverse primer.

[0053] For IAV, such as Figure 5 As shown in A to C, the optimal reverse primer was determined to be R207-236 (SEQ ID NO:23) using the forward primer F49-78 (SEQ ID NO:17), and then the optimal primer combination was determined to be the forward primer F57-86 (SEQ ID NO:18) and the reverse primer R207-236 (SEQ ID NO:23) using the reverse primer.

[0054] For RSV, such as Figure 6 As shown in A to C, the optimal reverse primer was determined to be R560-589 (SEQ ID NO:32) using the forward primer F446-475 (SEQ ID NO:29). Then, the optimal primer combination was determined to be the forward primer F420-449 (SEQ ID NO:27) and the reverse primer R560-589 (SEQ ID NO:32) using the reverse primer.

[0055] (2) Second round of screening

[0056] To obtain primer combinations with higher detection sensitivity, a second round of screening was conducted. Based on the optimal primer combinations for each molecular target obtained in the first round, four new forward primers and four new reverse primers were designed, and their specific sequences are shown in Table 2. The screening method and templates used were the same as in the first round.

[0057] Table 2. Sequence information of primers and exo probes used in the second round of screening.

[0058]

[0059]

[0060] FAM-dT: Thymidine nucleotide carrying FAM; TAMRA-dT: Thymidine nucleotide carrying TAMRA; ROX-dT: Thymidine nucleotide carrying ROX; BHQ1-dT: Thymidine nucleotide carrying BHQ1; BHQ2-dT: Thymidine nucleotide carrying BHQ2; THF: Tetrahydrofuran; C3-Spacer: C3-Spacer blocks elongation at the 3' end. Carboxyfluorescein (FAM); Tetramethylrhodamine (TAMRA); Rhodamine X (ROX).

[0061] For SARS-CoV-2, such as Figure 4 As shown in D to F, the optimal reverse primer was first determined to be R940-969 (SEQ ID NO:10) using the forward primer F804-833 (SEQ ID NO:6). Then, the new optimal primer combination was determined to be the forward primer F805-834 (SEQ ID NO:39) and the reverse primer R940-969 (SEQ ID NO:10) using the reverse primer.

[0062] For IAV, such as Figure 5 As shown in D to F, the optimal reverse primer was determined to be R207-236 (SEQ ID NO:23) using the forward primer F57-86 (SEQ ID NO:18). Then, the new optimal primer combination was determined using the reverse primer to be the forward primer F62-91 (SEQ ID NO:47) and the reverse primer R207-236 (SEQ ID NO:23).

[0063] For RSV, follow the same method, such as Figure 6 As shown in D to F, the optimal reverse primer was determined to be R559-589 (SEQ ID NO:58) using the forward primer F420-449 (SEQ ID NO:27). Then, the new optimal primer combination was determined using this reverse primer to be the forward primer F420-449 (SEQ ID NO:27) and the reverse primer R559-589 (SEQ ID NO:58).

[0064] Example 2: Performance Evaluation of Triple Real-Time RT-RAA Method for Detecting Respiratory Pathogens

[0065] 1. Specificity evaluation

[0066] (1) Experimental methods

[0067] Standard plasmids of 13 common respiratory pathogens (IAV, SARS-CoV-2, RSV, human parainfluenza virus type 1 (HPIV1), human parainfluenza virus type 3 (HPIV3), human metapneumovirus (HMPV), human adenovirus (HAdV), human rhinovirus (HRV), human coronavirus (HCoV), Chlamydia pneumoniae (C. pneumonia), Mycoplasma pneumoniae (M. pneumonia), influenza B virus (IBV), and human bocavirus type 1 (HBoV1)) were used as templates, with DEPC water as a negative control. The three primer combinations obtained in Example 1 (F805-834 (SEQ ID NO:39) and R940-969 (SEQ ID NO:10), F62-91 (SEQ ID NO:47) and R207-236 (SEQ ID NO:23), F420-449 (SEQ ID NO:27) and R559-589 (SEQ ID NO:10) were used. NO:58)), perform triple real-time RT-RAA detection.

[0068] The templates for each pathogen are as follows: SARS-CoV-2 uses pUC57-SARS-CoV-2-N; IAV uses pUC57-IAV-M; RSV uses pUC57-RSV-N; HPIV1 uses the HPIV1 M and N gene standard plasmid, denoted as pUC57-HPIV1-MN, obtained by ligating the M and N gene sequences of HPIV1 (NC_003461.1) into the pUC57 vector; HPIV3 uses the HPIV3 M gene standard plasmid, denoted as pUC57-HPIV3-M, obtained by ligating the M gene sequence of HPIV3 (NC_075446.1) into the pUC57 vector; HMPV uses the HMPV template. The standard plasmids for the M and N genes, denoted as pUC57-HMPV-MN, were obtained by ligating the M and N gene sequences of HMPV (NC_039199.1) into the pUC57 vector. The template for HADV was the HADV hexon gene standard plasmid, denoted as pUC57-HAdV-hexon, obtained by ligating the hexon gene sequence of HADV (LC795632.1) into the pUC57 vector. The template for HRV was the HRV polyprotein gene standard plasmid, denoted as pUC57-HRV-polyprotein, obtained by ligating the polyprotein gene sequence of HRV (JX193795.1) into the pUC57 vector. The template for HCoV was HCOV. The M gene standard plasmid, denoted as pUC57-HCOV-M, is obtained by ligating the M gene sequence of HCOV (NC_006577.2) into the pUC57 vector; the template for C. Pneumonia is the C. Pneumonia 16S RNA standard plasmid, denoted as pUC57-CP-16s, obtained by ligating the C. Pneumonia (NR_026527.1) 16S RNA gene sequence into the pUC57 vector; the template for M. Pneumonia is the M. Pneumonia P1 gene standard plasmid, denoted as pUC57-MP-P1, obtained by ligating the M. Pneumonia (MK330962.1) P1 gene sequence into the pUC57 vector; the template for IBV is IBV. The standard plasmid for the M gene, denoted as pUC57-IBV-M, is obtained by ligating the M gene sequence of IBV (PP699037.1) into the pUC57 vector; the template for HBoV1 is the standard plasmid for the HBoV1 VP2 gene, denoted as pUC57-HBoV1-VP2, which is obtained by ligating the VP2 gene sequence of HBoV1 (PP625021.11) into the pUC57 vector.

[0069] The triple real-time RT-RAA method uses a kit (#S002ZC) from Hangzhou Zhongce Biotechnology Co., Ltd. (China). The triple real-time RT-RAA system (50 μL / reaction) consists of the following components: Buffer A (25 μL); 3 forward primers, each 10 μM, in a 1:1:1 volume ratio, totaling 4.0 μL; 3 reverse primers, each 10 μM, in a 1:1:1 volume ratio, totaling 4.0 μL; 3 exo probes, each 10 μM, 0.6 μL each, totaling 1.8 μL; DEPC water, 8.7 μL; nucleic acid template, 4.0 μL; Buffer B (2.5 μL). The triple real-time RT-RAA reaction procedure is as follows: place the reaction tube in a QuantStudio real-time PCR System (Applied Biosystems, America) and incubate at 42°C for 20 minutes (1 cycle / min), monitoring the fluorescence signal in real time.

[0070] (2) Experimental Results

[0071] like Figure 7 As shown, the detection results for IAV, SARS-CoV-2, and RSV were all positive, while the detection results for HPIV1, HPIV3, HMPV, HADV, HRV, HCoV, C. pneumonia, M. pneumonia, IBV, HBoV1, and DEPC water were all negative. This indicates that the triple real-time RT-RAA method established in this invention has high specificity.

[0072] 2. Sensitivity Evaluation

[0073] (1) Experimental methods

[0074] pUC57-IAV-M was diluted with DEPC-treated water in five different gradients to obtain concentrations of 10-1. 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL and 10 0 Using standard plasmid dilution buffer (copies / μL) as a template and DEPC water as a negative control, triple real-time RT-RAA detection was performed using the primer combination F62-91 (SEQ ID NO:47) and R207-236 (SEQ ID NO:23) obtained in Example 1.

[0075] pUC57-SARS-CoV-2-N was diluted with DEPC water in five different dilutions to obtain concentrations of 10-1. 4 copies / μL, 10 3copies / μL, 10 2 copies / μL, 10 1 copies / μL, and 10 0 Using standard plasmid dilution buffer (copies / μL) as a template and DEPC water as a negative control, triple real-time RT-RAA detection was performed using the primer combination F805-834 (SEQ ID NO:39) and R940-969 (SEQ ID NO:10) obtained in Example 1.

[0076] pUC57-RSV-N was diluted with DEPC-treated water in five different gradients to obtain concentrations of 10-1. 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL and 10 0 Using standard plasmid dilution buffer (copies / μL) as a template and DEPC water as a negative control, triple real-time RT-RAA detection was performed using the primer combination F420-449 (SEQ ID NO:27) and R559-589 (SEQ ID NO:58) obtained in Example 1.

[0077] For comparison, triplet RT-qPCR was also performed using the same template. The triplet RT-qPCR method used the triplet RT-qPCR kit (#BNCC378336) from BeiNa Biotechnology Co., Ltd. (China). The triplet RT-qPCR system consisted of 23 μL Multiplex Master Mix and 2 μL template. The reaction tubes were placed in the QuantStudio Real-Time PCR System (Applied Biosystems), and the triplet RT-qPCR reaction conditions were as follows: initial 95℃ for 3 minutes; then 94℃ for 10 seconds and 56℃ for 30 seconds, for a total of 40 cycles, with real-time monitoring of the fluorescence signal. A CT value >35 was considered a negative result.

[0078] (2) Experimental Results

[0079] like Figure 8 As shown in A and B, the detection limits for IAV by both the triple real-time RT-RAA method and the triple RT-qPCR method are 10. 2 copies / μL. Similarly, such as... Figure 8 As shown in C-E, the detection limits for both tests for SARS-CoV-2 and RSV are 10. 2The number of copies / μL indicates that the triple real-time RT-RAA method established in this invention has high sensitivity.

[0080] Example 3: Performance Evaluation of Triple Real-Time RT-RAA Method for Detecting Clinical Samples

[0081] 1. Experimental Methods

[0082] For 58 clinical samples (2 bronchoalveolar lavage fluids, 15 cell culture isolates, and 41 oropharyngeal swabs), triple real-time RT-RAA and triple RT-qPCR were performed according to the method in Example 2. Kappa value, positive predictive value (PPV), and negative predictive value (NPV) were calculated based on the results of the two methods.

[0083] 2. Experimental Results

[0084] Table 3. Clinical sample detection results of triple RT-RAA and triple RT-qPCR

[0085]

[0086] As shown in Table 3, the sensitivity and specificity of the triple real-time RT-RAA detection were 100% (58 / 58) and 100.0% (58 / 58), respectively. The two detections showed very good correlation, with a Kappa value of 1 (Kappa=[58*(6+52)-(6*6+52*52)] / [58 2 The results showed that the triple real-time RT-RAA method established in this invention can meet the requirements of clinical testing. The P<0.001 value indicates that both PPV and NPV are 100%.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description and ideas, and it is neither necessary nor possible to exhaustively describe all implementation methods here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A primer-probe composition for use in the preparation of products for identifying respiratory pathogens based on RT-RAA, characterized in that, The primer-probe composition comprises primer-probe compositions 1 to 3; The primer-probe composition 1 comprises primer pair 4 and a probe with a nucleotide sequence as shown in SEQ ID NO:14; The primer-probe composition 2 comprises primer pair 5 and a probe with a nucleotide sequence as shown in SEQ ID NO:25; The primer-probe composition 3 comprises primer pair 6 and a probe with a nucleotide sequence as shown in SEQ ID NO:36; Primer pair 4 consists of primers with nucleotide sequences as shown in SEQ ID NO:39 and SEQ ID NO:10; Primer pair 5 consists of primers with nucleotide sequences as shown in SEQ ID NO:47 and SEQ ID NO:23; Primer pair 6 consists of primers with nucleotide sequences as shown in SEQ ID NO:27 and SEQ ID NO:

58.

2. An RT-RAA detection system for identifying respiratory pathogens, characterized in that, It comprises the primer-probe composition of claim 1 and the RAA reagent.

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