Molecular targets for identifying respiratory pathogens, primer composition, method and application thereof

Abstract

The invention discloses a group of molecular targets for identifying respiratory pathogens, a primer composition, a method and application thereof, the respiratory pathogens are novel coronavirus, influenza A virus and respiratory syncytial virus, and the nucleotide sequences of the molecular targets are respectively shown as SEQ ID NO: 1-3. The invention provides molecular targets, primers and probes for detecting the three respiratory tract pathogens, and establishes a triple real-time RT-RAA detection technology, so that the three respiratory tract infectious disease pathogens with similar clinical symptoms can be quickly distinguished and identified within 30 minutes, the operation threshold of the existing detection technology is reduced, and the detection efficiency is improved. The target of rapidly, simply and accurately distinguishing and identifying the three viruses is achieved, the requirement for early clinical diagnosis of infection is met, technical support is provided for precise medication, and the method has wide application value.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to a group of molecular targets, primer compositions, methods and applications thereof for identifying respiratory pathogens. Background Art

[0002] Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), influenza A virus (IAV) and respiratory syncytial virus (RSV) are common respiratory pathogens. The clinical symptoms of the diseases are very similar, and they all show symptoms such as fever, cough, and fatigue. It is difficult to distinguish them based on symptoms alone. Rapid identification and recognition of the three viruses in the early stage of infection and targeted drug administration are of great significance for improving the clinical treatment rate. Therefore, there is an urgent need for a detection technology that can quickly, specifically and accurately distinguish and identify the three viruses to meet the needs of early clinical diagnosis and treatment.

[0003] Reverse transcription polymerase chain reaction (RT-qPCR) technology is currently a commonly used technology for diagnosing respiratory pathogens. RT-qPCR detection methods for SARS-CoV-2, IAV, and RSV have been established in the prior art. However, the mutations caused by the adaptive evolution of these viruses often enable them to evade the detection primers and probes designed by the current RT-qPCR method, resulting in false negative results. In addition, the implementation of RT-qPCR requires well-trained technicians, expensive laboratory instruments, etc., which is undoubtedly a huge challenge in areas with limited medical resources but relatively more prone to infectious diseases. Therefore, there is an urgent need to develop a rapid detection technology that is simple to operate and does not require special equipment.

[0004] Reverse transcription-recombinase-aided amplification (RT-RAA) is a new isothermal in vitro nucleic acid amplification technology. It first uses reverse transcriptase to reverse transcribe RNA samples into cDNA, and then uses recombinase combined with primers and probes to mediate strand displacement reactions, thereby achieving real-time amplification detection. The detection can be completed within 30 minutes at 37-42°C. It has the characteristics of high sensitivity, strong specificity, short reaction time, simple operation, intuitive result judgment, and suitability for rapid on-site diagnosis. It has been applied to the single-plex detection of various pathogenic microorganisms. However, there are currently few reports on multiplex RT-RAA detection technology for the simultaneous detection of SARS-CoV-2, IAV, and RSV. This is mainly because the target sequence length of the target gene detected by RAA technology is relatively short, while the genome sequences of these three pathogens are highly similar. RAA amplification primers designed based on existing commonly used molecular targets are prone to nonspecific amplification, affecting detection accuracy. In addition, as the number of detection targets increases, the optimization and balance of the multiplex RAA detection system becomes more complicated, and issues such as the interaction between primer pairs, detection and differentiation of amplification products, etc. need to be considered. Summary of the invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a group of molecular targets, primer compositions, methods and applications thereof for identifying respiratory pathogens.

[0006] The first object of the present invention is to provide a set of molecular targets for identifying respiratory pathogens.

[0007] The second object of the present invention is to provide the use of a reagent for detecting the molecular target in the preparation of a product for identifying respiratory pathogens.

[0008] The third object of the present invention is to provide primers for detecting the molecular target.

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

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

[0011] The sixth object of the present invention is to provide the use of the primer, the primer combination or the primer-probe combination in the preparation of a product for identifying respiratory pathogens.

[0012] A seventh object of the present invention is to provide a method for identifying respiratory pathogens for non-disease treatment and diagnosis purposes.

[0013] The eighth object of the present invention is to provide a RT-RAA detection system for identifying respiratory pathogens.

[0014] In order to achieve the above object, the present invention is implemented by the following scheme:

[0015] Based on the newly discovered molecular targets, the present invention established a triple real-time RT-RAA detection method to quickly and accurately identify three respiratory pathogens with similar clinical symptoms - influenza A virus, new coronavirus and respiratory syncytial virus.

[0016] A group of molecular targets for identifying respiratory pathogens, wherein the respiratory pathogens are novel coronavirus, influenza A virus and respiratory syncytial virus, and the nucleotide sequences of the molecular targets are shown in SEQ ID NOs: 1 to 3, respectively. Among them, the nucleotide sequence of the target sequence of the novel coronavirus is shown in SEQ ID NO: 1; the nucleotide sequence of the target sequence of influenza A virus is shown in SEQ ID NO: 2; and the nucleotide sequence of the target sequence of respiratory syncytial virus is shown in SEQ ID NO: 3.

[0017] The use of the molecular target reagent in the preparation of products for identifying respiratory pathogens should also be within the scope of protection of the present invention.

[0018] Primers for detecting the molecular target, the nucleotide sequences of the primers are shown in SEQ ID NOs: 4-13, SEQ ID NOs: 15-24, SEQ ID NOs: 26-35 and SEQ ID NOs: 37-59. Among them, the primers with nucleotide sequences as shown in SEQ ID NOs: 4-13 and SEQ ID NOs: 37-44 detect the new coronavirus; the primers with nucleotide sequences as shown in SEQ ID NOs: 15-24 and SEQ ID NOs: 45-51 detect influenza A virus; the primers with nucleotide sequences as shown in SEQ ID NOs: 26-35 and SEQ ID NOs: 52-59 detect respiratory syncytial virus.

[0019] The primer composition for detecting the molecular target comprises primer pairs 1 to 6; the primer pair 1 is a primer having a nucleotide sequence as shown in SEQ ID NO: 6 and SEQ ID NO: 10; the primer pair 2 is a primer having a nucleotide sequence as shown in SEQ ID NO: 18 and SEQ ID NO: 23; the primer pair 3 is a primer having a nucleotide sequence as shown in SEQ ID NO: 27 and SEQ ID NO: 32; the primer pair 4 is a primer having a nucleotide sequence as shown in SEQ ID NO: 39 and SEQ ID NO: 10; the primer pair 5 is a primer having a nucleotide sequence as shown in SEQ ID NO: 47 and SEQ ID NO: 23; the primer pair 6 is a primer having a nucleotide sequence as shown in SEQ ID NO: 27 and SEQ ID NO: 58. Among them, the primer pair 1 and the primer pair 4 detect the new coronavirus; the primer pair 2 and the primer pair 5 detect influenza A virus; the primer pair 3 and the primer pair 6 detect respiratory syncytial virus.

[0020] The primer-probe composition for detecting the molecular target comprises a primer and a probe, wherein the primer is a primer 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 sequence of the probe is as shown in SEQ ID NO: 14, 25 and 36. Among them, the probe with a nucleotide sequence as shown in SEQ ID NO: 14 detects the new coronavirus; the probe with a nucleotide sequence as shown in SEQ ID NO: 25 detects influenza A virus; and the probe with a nucleotide sequence as shown in SEQ ID NO: 36 detects respiratory syncytial virus.

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

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

[0024] Further preferably, the probes in each group of the primer-probe combinations 1 to 3 are provided with different fluorescent groups, and the fluorescent groups are TAMRA, FAM or ROX.

[0025] The use of the primer, the primer combination or the primer-probe combination in the preparation of products for identifying respiratory pathogens should also be within the protection scope of the present invention.

[0026] A method for identifying respiratory pathogens for the purpose of non-disease treatment and diagnosis, using the primer, the primer combination or the primer-probe combination, and the RT-RAA method for detection.

[0027] An RT-RAA detection system for identifying respiratory pathogens comprises the primer, the primer combination or the primer-probe combination and a RAA reagent.

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

[0029] The present invention provides molecular targets, primers and probes for detecting three respiratory pathogens, and establishes a triple real-time RT-RAA detection technology, which can achieve rapid differentiation and identification of three respiratory infectious disease pathogens with similar clinical symptoms within 30 minutes, lowering the operating threshold of existing detection technologies, achieving the goal of rapid, simple and accurate differentiation and identification of three viruses, meeting the needs of early clinical diagnosis of infection, providing technical support for precision drug use, and having broad application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The positions of RT-RAA primers and exo probes on the M gene sequences of different IAV strains; the parts covered by the boxes are the corresponding matching regions of the forward primer, exo probe and reverse primer respectively; the solid triangles and hollow triangles mark the two T residues in the fluorescent group and quenching group of the exo probe respectively; the arrow marks tetrahydrofuran.

[0031] Figure 2The positions of RT-RAA primers and exo probes on the N gene sequences of different SARS-CoV-2 strains; the parts covered by the boxes are the corresponding matching regions of the forward primer, exo probe and reverse primer, respectively; the solid triangles and hollow triangles mark the two T residues in the fluorescent group and quenching group of the exo probe, respectively; the arrow marks tetrahydrofuran.

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

[0033] Figure 4 Primers for triple real-time RT-RAA detection of SARS-CoV-2 are determined; A is a schematic diagram of the principle of the first round of primer screening. In the primer name, the number indicates 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 primer; C is the first round screening result of the forward primer; 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 primer; F is the second round screening result of the forward primer.

[0034] Figure 5 Primers for triple real-time RT-RAA detection of IAV are determined; A is a schematic diagram of the principle of the first round of primer screening, in the primer name, the number indicates the position in the M gene of IAV (GenBank accession number: MN570352.1); B is the first round screening result of the reverse primer; C is the first round screening result of the forward primer; 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 primer, and F is the second round screening result of the forward primer.

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

[0036] Figure 7These are the specificity evaluation results of the triple real-time RT-RAA method. 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 sensitivity evaluation results of the triple real-time RT-RAA method; curves 1 to 5 correspond to 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL and 10 0 copies / μL of standard plasmid dilution, curve 6 corresponds to the negative control; A and B are the results of triple real-time RT-RAA and triple RT-qPCR detection of IAV, respectively; C and D are the results of triple real-time RT-RAA and triple RT-qPCR detection of SARS-CoV-2, respectively; E and F are the results of triple real-time RT-RAA and triple RT-qPCR detection of RSV, respectively. DETAILED DESCRIPTION

[0038] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

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

[0040] 1. Determination of molecular targets

[0041] 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, a molecular target for detecting SARS-CoV-2 (the nucleotide sequence is shown in SEQ ID NO: 1), a molecular target for IAV (the nucleotide sequence is shown in SEQ ID NO: 2), and a molecular target for RSV (the nucleotide sequence is shown in SEQ ID NO: 3) were obtained.

[0042] 2. Design of primers and probes and determination of the optimal primer combination

[0043] SnapGene software (version 3.6.2022) was used to design primers and exo probes for the three molecular targets obtained, and the primers and exo probes were designed according to the TwistAmp TM The best primer combination was screened according to the amplification guidelines.

[0044] (1) First round of screening

[0045] Five forward primers and five reverse primers were designed for each 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 in the first round of screening

[0047]

[0048]

[0049] FAM-dT, thymine nucleotide carries FAM; TAMRA-dT, thymine nucleotide carries TAMRA; ROX-dT, thymine nucleotide carries ROX; BHQ1-dT, thymine nucleotide carries BHQ1; BHQ2-dT, thymine nucleotide carries BHQ2; THF, tetrahydrofuran; C3-Spacer, C3-Spacer blocks elongation at the 3' end. Carboxyfluorescein (FAM); Tetramethylrhodamine (TAMRA); Rhodamine X (ROX).

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

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

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

[0053] For IAV, Figure 5 As shown in A to C in FIG, the best reverse primer is determined to be R207-236 (SEQ ID NO: 23) using the forward primer F49-78 (SEQ ID NO: 17), and then the best primer combination is 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, Figure 6 As shown in A to C in FIG, the best reverse primer is determined to be R560-589 (SEQ ID NO: 32) using the forward primer F446-475 (SEQ ID NO: 29), and then the best primer combination is 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] In order to obtain a primer combination with higher detection sensitivity, a second round of screening was continued. Four new forward primers and four new reverse primers were redesigned based on the best primer combination for each molecular target obtained in the first round. The specific sequences are shown in Table 2. The screening method and template used were the same as those in the first round.

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

[0058]

[0059]

[0060] FAM-dT, thymine nucleotide carries FAM; TAMRA-dT, thymine nucleotide carries TAMRA; ROX-dT, thymine nucleotide carries ROX; BHQ1-dT, thymine nucleotide carries BHQ1; BHQ2-dT, thymine nucleotide carries 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, Figure 4 As shown in D to F in the figure, the forward primer F804-833 (SEQ ID NO: 6) is first used to determine that the best reverse primer is R940-969 (SEQ ID NO: 10), and then the reverse primer is used to determine a new best primer combination as forward primer F805-834 (SEQ ID NO: 39) and reverse primer R940-969 (SEQ ID NO: 10).

[0062] For IAV Figure 5 As shown in D to F in the figure, the best reverse primer is determined to be R207-236 (SEQ ID NO: 23) using the forward primer F57-86 (SEQ ID NO: 18), and then the reverse primer is used to determine a new best primer combination as 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 approach as Figure 6 As shown in D to F in the figure, the best reverse primer is determined to be R559-589 (SEQ ID NO: 58) using the forward primer F420-449 (SEQ ID NO: 27), and then the reverse primer is used to determine a new best primer combination as 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] The 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, DEPC water was used as a negative control, and 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: 29) were used as primers. NO:58)), triple real-time RT-RAA detection was performed.

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

[0069] The triple real-time RT-RAA method used a kit (#S002ZC) from Hangzhou Zhongce Biotechnology Co., Ltd. (China). The triple real-time RT-RAA system (50 μL / reaction) consisted of the following components: buffer A (25 μL); 3 forward primers, each at a concentration of 10 μM, in a volume ratio of 1:1:1, for a total of 4.0 μL; 3 reverse primers, each at a concentration of 10 μM, in a volume ratio of 1:1:1, for a total of 4.0 μL; 3 exo probes, each at a concentration of 10 μM, 0.6 μL each, for a total of 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 was as follows: the reaction tube was placed in a QuantStudio real-time PCR System (Applied Biosystems, America), incubated at 42°C for 20 minutes (1 cycle / min), and the fluorescence signal was monitored in real time.

[0070] (2) Experimental results

[0071] like Figure 7 As shown, the detection results of IAV, SARS-CoV-2 and RSV were all positive, while the detection results of HPIV1, HPIV3, HMPV, HAdV, HRV, HCoV, C.Pneumonia, M.Pneumonia, IBV, HBoV1 and DEPC water were all negative, indicating that the triple real-time RT-RAA method established by the present invention has strong specificity.

[0072] 2. Sensitivity evaluation

[0073] (1) Experimental methods

[0074] Use DEPC water to dilute pUC57-IAV-M, dilute it 5 times, and get the concentration of 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL and 10 0 A standard plasmid dilution of 100 copies / μL was used as a template, DEPC water was used as a negative control, and the primer combination F62-91 (SEQ ID NO: 47) and R207-236 (SEQ ID NO: 23) obtained in Example 1 was used for triple real-time RT-RAA detection.

[0075] Use DEPC water to dilute pUC57-SARS-CoV-2-N, dilute five times to obtain concentrations of 10 4 copies / μL, 10 3copies / μL, 10 2 copies / μL, 10 1 copies / μL, and 10 0 A standard plasmid dilution of 100 copies / μL was used as a template, DEPC water was used as a negative control, and the primer combination F805-834 (SEQ ID NO: 39) and R940-969 (SEQ ID NO: 10) obtained in Example 1 was used for triple real-time RT-RAA detection.

[0076] Use DEPC water to dilute pUC57-RSV-N, dilute five times, and obtain concentrations of 10 4 copies / μL, 10 3 copies / μL, 10 2 copies / μL, 10 1 copies / μL and 10 0 A standard plasmid dilution of 100 copies / μL was used as a template, DEPC water was used as a negative control, and the primer combination F420-449 (SEQ ID NO: 27) and R559-589 (SEQ ID NO: 58) obtained in Example 1 was used for triple real-time RT-RAA detection.

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

[0078] (2) Experimental results

[0079] like Figure 8 As shown in A and B in Figure 1, the detection limit of the triple real-time RT-RAA method and the triple RT-qPCR method for IAV was 10 2 copies / μL. Similarly, Figure 8 As shown in Figures C to E, the detection limits of both tests for SARS-CoV-2 and RSV are both 10 2This indicates that the triple real-time RT-RAA method established in the present 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 detection and triple RT-qPCR detection were performed according to the method in Example 2, and the Kappa value, positive predictive value (PPV), and negative predictive value (NPV) were calculated based on the detection results of the two methods.

[0083] 2. Experimental results

[0084] Table 3 Clinical sample test 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 assay were 100% (58 / 58) and 100.0% (58 / 58), respectively. The two assays showed very good correlation, with a Kappa value of 1 (Kappa = [58*(6+52)-(6*6+52*52)] / [58 2 )-(6*6+52*52)]), P<0.001, PPV and NPV were both 100%. This indicates that the triple real-time RT-RAA method established in the present invention can meet the requirements of clinical detection.

[0087] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A set of molecular targets for identifying respiratory pathogens, characterized in that: The respiratory pathogens are novel coronavirus, influenza A virus and respiratory syncytial virus, and the nucleotide sequences of the molecular targets are shown in SEQ ID NOs: 1 to 3, respectively.

2. Use of a reagent for detecting the molecular target of claim 1 in the preparation of a product for identifying respiratory pathogens.

3. A primer for detecting the molecular target according to claim 1, characterized in that: The nucleotide sequences of the primers are shown in SEQ ID NOs: 4-13, SEQ ID NOs: 15-24, SEQ ID NOs: 26-35 and SEQ ID NOs: 37-59.

4. A primer composition for detecting the molecular target according to claim 1, characterized in that: Contains primer pairs 1 to 6; The primer pair 1 is a primer having nucleotide sequences as shown in SEQ ID NO: 6 and SEQ ID NO: 10; The primer pair 2 is a primer having nucleotide sequences as shown in SEQ ID NO: 18 and SEQ ID NO: 23; The primer pair 3 is a primer having nucleotide sequences as shown in SEQ ID NO: 27 and SEQ ID NO: 32; The primer pair 4 is a primer having a nucleotide sequence as shown in SEQ ID NO:39 and SEQ ID NO:10; The primer pair 5 is a primer having nucleotide sequences as shown in SEQ ID NO:47 and SEQ ID NO:23; The primer pair 6 is a primer having nucleotide sequences as shown in SEQ ID NO: 27 and SEQ ID NO:

58.

5. A primer-probe composition for detecting the molecular target according to claim 1, characterized in that: The method comprises a primer and a probe, wherein the primer is the primer according to claim 3 or the primer combination according to claim 4.

6. The primer-probe combination according to claim 5, characterized in that: The nucleotide sequences of the probes are shown in SEQ ID NOs: 14, 25 and 36.

7. The primer-probe composition according to claim 6, characterized in that: The primer is the primer composition according to claim 4, and the primer-probe composition comprises primer-probe compositions 1 to 3; The primer-probe composition 1 comprises primer pair 1 or primer pair 4, and a probe having a nucleotide sequence as shown in SEQ ID NO: 14; The primer-probe combination 2 comprises primer pair 2 or primer pair 5, and a probe having a nucleotide sequence as shown in SEQ ID NO: 25; The primer-probe combination 3 comprises primer pair 3 or primer pair 6, and a probe having a nucleotide sequence as shown in SEQ ID NO:

36.

8. Use of the primer according to claim 3, the primer combination according to claim 4, or the primer-probe combination according to any one of claims 5 to 8 in the preparation of a product for identifying respiratory pathogens.

9. A method for identifying respiratory pathogens for non-disease treatment and diagnosis purposes, characterized in that: The detection is performed using the primers according to claim 3, the primer combination according to claim 4, or the primer-probe combination according to any one of claims 5 to 8, and the RT-RAA method.

10. An RT-RAA detection system for identifying respiratory pathogens, characterized in that: The method comprises the primer according to claim 3, the primer combination according to claim 4 or the primer-probe combination according to any one of claims 5 to 8 and a RAA reagent.

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