Method for realizing multiple detection by selecting different to-be-detected target combinations in enzyme digestion probe isothermal detection method and kit thereof

Abstract

The invention provides a method for realizing multiple detection by selecting different to-be-detected target combinations in an enzyme digestion probe isothermal detection method and a kit thereof, and the method comprises the following steps: respectively designing a corresponding enzyme digestion probe isothermal detection probe RNHP and a primer aiming at each to-be-detected target; and comparing the first fluorescent probe RNHP of the first target to be detected with the second fluorescent probe RNHP of the second target to be detected and all LAMP isothermal amplification primers of the second target to be detected, and combining if the number of continuous complementary paired basic groups of each heterodimer is less than 6. The invention provides a method for controlling the number of heterodimer connection bases of primers and probes between targets. When the number of heterodimer connection bases between two targets or between three targets is 1t; the target combination shows a remarkable and excellent combination success rate when being used for detecting 6 bp, so that the success rate of isothermal double / triple system combination can be increased, and the time of combination verification can be greatly shortened.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of molecular biology, in particular to a method for realizing multiplex detection by selecting different target combinations in an isothermal detection method of enzyme-cleavage probes and a kit thereof. BACKGROUND

[0002] Respiratory tract infection diseases are high-incidence clinical diseases, and various viral pathogens can cause serious respiratory diseases. The high-incidence pathogens include 14 types, including the novel coronavirus, adenovirus type 3 / 7, adenovirus type 55, respiratory syncytial virus, influenza A virus, influenza B virus, Epstein-Barr virus, rhinovirus, metapneumovirus 1B, metapneumovirus A2b, cytomegalovirus, parainfluenza virus type 1, parainfluenza virus type 2, and parainfluenza virus type 3. The clinical cases of respiratory tract infection viruses are significantly high in incidence. Therefore, realizing rapid and sensitive detection of viral pathogens provides an important basis for accurate diagnosis by doctors and is of great significance for disease diagnosis and treatment of patients.

[0003] At present, the conventional molecular detection technology for detecting viruses in clinical detection, such as fluorescence quantitative PCR, can diagnose the type of pathogen by detecting nucleic acids in the sample, and the detection result can be obtained within 1-2 hours. However, this method requires precise instruments and has high economic cost. The isothermal detection technology does not require temperature variation steps, has low precision requirement for instruments, and has short detection time of 20-30 minutes, so it has low time cost and economic cost, and is suitable for diagnosis of viral respiratory diseases in primary medical institutions.

[0004] CN109750091B discloses an isothermal detection method using enzyme digestion probes. The method includes the following steps: Step 1: Design specific isothermal amplification primers and fluorescent probes (RNHP) for each target nucleic acid sequence. The RNHP is labeled with different fluorescent markers for each target nucleic acid sequence, enabling differentiation between different target nucleic acid sequences in different fluorescence signal channels. The RNHP contains at least one RNA base. A fluorescent group is labeled on the probe base on the left side of the RNA base near the 5' end of the probe, and a quenching group is labeled on the probe base on the side of the RNA base near the 3' end of the probe. Preferably, the at least one RNA base is a ligand. Step 1: Continued RNA bases; Step 2: In the presence of ribonuclease RNase H, each target nucleic acid sequence is amplified isothermally under the action of nucleic acid polymerase, and each fluorescent probe RNHP binds to the corresponding target nucleic acid sequence to form a probe-target nucleic acid hybrid double strand; The ribonuclease RNase H cuts the RNA bases in the probe-target nucleic acid hybrid double strand, so that the probe fragment containing the quenching group on the right side of the RNA base is released, while the fragment containing the fluorescent group on the left side of the RNA base remains to form a hybrid chain and the fluorescent group emits fluorescence; and Step 3: The target nucleic acid sequence is detected by the hybrid chain products with different fluorescent labels formed by the target nucleic acid sequence. This method, based on isothermal amplification technology, achieves real-time multiplex isothermal detection by introducing a special modified primer RNHP and binding it to RNase H2. It can detect both DNA and RNA nucleic acids. Compared to existing real-time isothermal amplification methods using molecular beacons or other labeled probes: in the presence of target nucleic acids, RNHP, being a linear probe with a simple labeling group, binds more easily to the target nucleic acid, thus improving sensitivity. Compared to single-weighted isothermal amplification methods: it allows for the addition of internal controls, making the detection results more effective and reliable, and the result interpretation more objective. The entire process is performed in a closed tube to avoid amplification contamination.

[0005] However, current LAMP isothermal detection technology faces technical challenges in constructing single-tube multiplex detection systems, hindering the rapid detection of various viral pathogens. Therefore, developing a method or kit based on isothermal detection technology for the rapid and sensitive detection of viral pathogens causing respiratory infections is of significant guiding importance for achieving rapid and accurate diagnosis of the types of pathogens infecting patients. Summary of the Invention

[0006] To address the aforementioned issues, this invention provides a method for multiplex detection using isothermal enzyme digestion probes, employing different combinations of target molecules. This method designs specific isothermal amplification primers and fluorescent probes (RNHPs) for each target molecule. The RNHPs for each target molecule are labeled with different fluorescent molecules, enabling differentiation between different targets in different fluorescence signal channels. Each RNHP contains at least one RNA base. A fluorescent group is labeled on the probe base to the left of the RNA base near the 5' end of the probe, and a quenching group is labeled on the probe base to the right of the RNA base near the 3' end of the probe. The RNHP is 16-45 bp in length, and the distance between the fluorescent group and the quenching group is 5-15 bp. The G+C content in the probe fragment to the left of the RNA base is 40%-60%. The isothermal amplification is LAMP isothermal amplification. The method includes specific isothermal amplification primers and fluorescent probes (RNHPs) for each target molecule. For each target to be tested, corresponding enzyme digestion probes, isothermal detection probes (RNHP), and primers are designed. Then, the first fluorescent probe (RNHP) of the first target to be tested is compared with the second fluorescent probe (RNHP) of the second target to be tested and all primers for isothermal amplification of the second target to be tested (LAMP). If the heterodimers formed by the first fluorescent probe (RNHP) with the second fluorescent probe (RNHP) of the second target to be tested and all primers for isothermal amplification of the second target to be tested (LAMP) have a number of consecutive complementary base pairs of less than 6 (excluding 6), and if the heterodimers formed by the second fluorescent probe (RNHP) with the first fluorescent probe (RNHP) of the first target to be tested and all primers for isothermal amplification of the first target to be tested (LAMP) have a number of consecutive complementary base pairs of less than 6 (excluding 6), then the first target to be tested and the second target to be tested can be combined to achieve multiplex detection in the enzyme digestion probe isothermal detection method.

[0007] In one embodiment, the present invention provides a multiplex detection kit for different combinations of target molecules in an enzyme digestion probe isothermal detection method. The kit includes specific isothermal amplification primers and fluorescent probes (RNHP) designed for each target molecule. Each target molecule's RNHP is labeled with a different fluorescent group, allowing different targets to be distinguished in different fluorescence signal channels. The RNHP contains at least one RNA base, with a fluorescent group labeled on the probe base to the left of the RNA base near the 5' end and a quenching group labeled on the probe base to the right of the RNA base near the 3' end. The RNHP is 16-45 bp in length, and the distance between the fluorescent group and the quenching group on the RNHP is 5-15 bp. The G+C content in the probe fragment to the left of the RNA base is 40%-60%. The isothermal amplification described is LAMP isothermal amplification. The kit includes corresponding enzyme digestion probes, isothermal detection probes (RNHP), and primers designed for each target. The first fluorescent probe (RNHP) of the first target is compared with the second fluorescent probe (RNHP) of the second target and all primers for LAMP isothermal amplification of the second target. The first fluorescent probe (RNHP) forms corresponding heterodimers with the second fluorescent probe (RNHP) of the second target and all primers for LAMP isothermal amplification of the second target, respectively. The number of consecutive complementary base pairs in each heterodimer is less than 6, excluding 6. At the same time, the second fluorescent probe (RNHP) forms corresponding heterodimers with the first fluorescent probe (RNHP) of the first target and all primers for LAMP isothermal amplification of the first target, respectively. The number of consecutive complementary base pairs in each heterodimer is less than 6, excluding 6.

[0008] In one embodiment, the present invention provides a kit for isothermal detection of 14 respiratory infectious viruses using enzyme digestion probes. The kit effectively combines 14 viral targets into 7 multiplex reaction solutions: Reaction solution 1 detects novel coronavirus and adenovirus type 3 / 7; Reaction solution 2 detects respiratory syncytial virus and parainfluenza virus type 2; Reaction solution 3 detects influenza A virus and Epstein-Barr virus; Reaction solution 4 detects rhinovirus A1 and adenovirus type 55; Reaction solution 5 detects cytomegalovirus and influenza B virus; Reaction solution 6 detects metapneumovirus B1 and parainfluenza virus type 1; Reaction solution 7 detects metapneumovirus A2b, parainfluenza virus type 3, and an internal control.

[0009] In one embodiment, the reaction solution 1 includes LAMP isothermal amplification primers and fluorescent probe RNHP for detecting the novel coronavirus, namely SEQ ID No. 1-SEQ ID No. 6 and SEQ ID No. 7, respectively, and LAMP isothermal amplification primers and fluorescent probe RNHP for detecting adenovirus type 3 / 7, namely SEQ ID No. 15-SEQ ID No. 19 and SEQ ID No. 20.

[0010] In one embodiment, the reaction solution 2 includes LAMP isothermal amplification primers and fluorescent probe RNHP for detecting respiratory syncytial virus (RSV) as SEQ ID No. 8-SEQ ID No. 13 and SEQ ID No. 14, and LAMP isothermal amplification primers and fluorescent probe RNHP for detecting parainfluenza virus type 2 as SEQ ID No. 79-SEQ ID No. 83 and SEQ ID No. 84.

[0011] In one embodiment, the reaction solution 3 includes LAMP isothermal amplification primers and fluorescent probe RNHP for detecting influenza A virus, namely SEQ ID No. 27-SEQ ID No. 33 and SEQ ID No. 34, and LAMP isothermal amplification primers and fluorescent probe RNHP for detecting EB virus, namely SEQ ID No. 41-SEQ ID No. 45 and SEQ ID No. 46.

[0012] In one embodiment, the reaction solution 4 includes LAMP isothermal amplification primers and fluorescent probe RNHP for detecting rhinovirus A1 as SEQ ID No. 47-SEQ ID No. 52 and SEQ ID No. 53, and LAMP isothermal amplification primers and fluorescent probe RNHP for detecting adenovirus 55 as SEQ ID No. 21-SEQ ID No. 25 and SEQ ID No. 26.

[0013] In one embodiment, the reaction solution 5 includes LAMP isothermal amplification primers and fluorescent probe RNHP for cytomegalovirus (SEQ ID No. 54-SEQ ID No. 59, SEQ ID No. 60), and LAMP isothermal amplification primers and fluorescent probe RNHP for detecting adenovirus B (SEQ ID No. 35-SEQ ID No. 39, SEQ ID No. 40).

[0014] In one embodiment, the reaction solution 6 includes LAMP isothermal amplification primers and fluorescent probe RNHP for detecting metapneumovirus B1 as SEQ ID No. 61-SEQ ID No. 64 and SEQ ID No. 65, and LAMP isothermal amplification primers and fluorescent probe RNHP for detecting parainfluenza virus type 1 as SEQ ID No. 72-SEQ ID No. 77 and SEQ ID No. 78.

[0015] In one embodiment, the reaction solution 7 includes LAMP isothermal amplification primers and fluorescent probe RNHP for detecting metapneumovirus A2b (SEQ ID No. 66-SEQ ID No. 70, SEQ ID No. 71), LAMP isothermal amplification primers and fluorescent probe RNHP for detecting parainfluenza virus type 3 (SEQ ID No. 85-SEQ ID No. 89, SEQ ID No. 90), and LAMP isothermal amplification primers and fluorescent probe RNHP for detecting the internal control (SEQ ID No. 91-SEQ ID No. 95, SEQ ID No. 96).

[0016] This invention establishes a method or kit for rapid and sensitive detection of viral pathogens causing respiratory infections based on isothermal detection technology, which has important guiding significance for achieving rapid and accurate diagnosis of the type of pathogen infecting patients. This invention provides a method for controlling the number of bases linked in heterodimers between targets. When the number of bases linked in heterodimers between two or three targets is <6 bp, the target combinations exhibit a significantly superior success rate, which can improve the success rate of isothermal two / triple systems and greatly reduce the time for combination validation. Based on this, seven isothermal multiplex reaction solutions for detecting 14 viral nucleic acids were established. Detailed Implementation

[0017] To enable those skilled in the art to better understand the technical solutions in this application, the present invention will be further described below with reference to embodiments. Obviously, the embodiments are merely examples and should not be construed as limiting the present invention. Based on the embodiments in this application, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of this application. The experimental methods used in this invention are all conventional methods, and the experimental equipment, materials, reagents, etc. used can all be obtained commercially.

[0018] Example 1: Verification of the influence of the number of bases linked in the heterodimer of primers and probes between multiple targets on the feasibility of multiple combinations.

[0019] I. Sequence information of the optimal single primers and probes for each detection target screened in this study This invention analyzes the gene sequences of pathogens and internal reference genes of viruses such as novel coronavirus (NCP), respiratory syncytial virus (RSV), adenovirus type 3 (HADV3), adenovirus type 55 (HADV55), influenza A virus (IVA), influenza B virus (IVB), Epstein-Barr virus (EBV), rhinovirus (HRV), cytomegalovirus (HCMV), metapneumovirus 1B (HMPV1b), metapneumovirus A2b (HMPVA2b), parainfluenza virus type 1 (HPIV1), parainfluenza virus type 2 (HPIV2), and parainfluenza virus type 3 (HPIV3). Based on the basic principles of primer and probe design, primer and probe combinations were designed using design software. The optimal primer and probe sequences for each pathogen target and internal reference were screened. The sequence information of the single optimal primer and probe for each target is shown in Tables 1 and 2.

[0020] Table 1. Sequence information of 14 viral diseases and their internal reference primers and probes.

[0021] Table 2. Sequence information of 14 viral diseases and their internal reference primers and probes.

[0022] II. Verification of the number of bases required for heterodimer linkage between primers and probes in multi-target combinations. In isothermal amplification systems using enzyme-digested probes, because each target has as many as 5-7 primers, amplification inhibition often occurs due to non-specific binding between the primers and probes of each target when performing single-tube duplex or multiplex combinations, resulting in duplex or multiplex combination failure. In this study, before performing multiplex combinations on each target, we compared the optimal probes for each target identified in Table 1 with primers and probes for other targets. After comparison, by controlling the number of consecutive bases in the heterodimers that appeared, we first verified the duplex combinations of 14 viral targets, and then verified the effective duplex combinations with the internal control in triplex combinations.

[0023] The templates used in this validation included: nucleic acids extracted from clinical samples infected with 14 viruses, standard plasmid dilutions of 5 copies / μL and 10 copies / μL, and human genomic DNA.

[0024] In this embodiment, six double isothermal reaction solutions and one triple isothermal reaction solution were finally established ("NCP + HADV3", "RSV + HPIV2", "HRV + HADV55", "IVB + HCMV", "HMPV1b + HPIV1", "HMPVA2b + HPIV3 + WNC", "IVA + EB"). The specific establishment process is shown below.

[0025] 1. Validation and establishment of the double isothermal detection reaction solution 1) The primers and probes for 14 viral targets were analyzed by heterodimer analysis. The number of heterodimer linkages between primers and probes for each target is shown in Tables 3-16.

[0026] Table 3. Base number information of heterodimers formed between the COVID-19 probe and other target primers / probes.

[0027] Table 4. Base number information for heterodimer linkages formed between the respiratory syncytial probe and other target primers / probes.

[0028] Table 5. Base number information for heterodimer linkages formed between adenovirus type 3 / 7 probes and other target primers / probes.

[0029] Table 6. Base number information for heterodimer linkages formed between adenovirus type 55 probe and other target primers / probes.

[0030] Table 7. Base number information of heterodimers formed between influenza A probes and other target primers / probes.

[0031] Table 8. Information on the number of bases linked to the heterodimers formed between the acetylene probe and other target primers / probes.

[0032] Table 9 shows the number of bases linked to the heterodimers formed between the EB probe and other target primers / probes.

[0033] Table 10. Base number information for heterodimers formed between rhinovirus probes and other target primers / probes.

[0034] Table 11 Information on the number of bases linked to the heterodimers formed between the giant cell probe and other target primers / probes.

[0035] Table 12. Base number information of heterodimers formed between parainfluenza type 1 probes and other target primers / probes.

[0036] Table 13 Information on the number of bases linked to heterodimers formed between parainfluenza type 2 probes and other target primers / probes.

[0037] Table 14. Base number information of heterodimers formed between parainfluenza type 3 probes and other target primers / probes.

[0038] Table 15 Information on the number of bases linked to heterodimers formed between the metapneumovirus A2b probe and other target primers / probes.

[0039] Table 16 Information on the number of bases linked to heterodimers formed between the pneumovirus B1 probe and other target primers / probes.

[0040] 2) Enzyme digestion probe isothermal dual detection system and detection procedure (Table 17) Table 17 Components and Detection Procedure of the Isothermal Detection System for Enzyme Digestion Probes in a Dual System

[0041] 3) Experimental verification of the dual-system target combination Based on the number of linked bases in the heterodimer, the verification was divided into two categories for comparative verification: Category 1: target combinations with <6 bp linked bases in the primer-probe heterodimer; Category 2: target combinations with ≥6 bp linked bases in the primer-probe heterodimer.

[0042] The first category of target combinations includes: “NCP+HRV”, “NCP + HADV3”, “RSV + HPIV2”, “RSV+HRV”, “IVA + HMPVA2b”, “HRV + HADV55”, “HRV + HCMV”, “IVB + HCMV”, “HMPVA2b +HPIV3”, and “HMPV1b+HPIV1”.

[0043] The second category of target combinations includes: “NCP+RSV”, “NCP+HCMV”, “RSV + IVB”, “RSV+ HMPV1b”, “IVA + HCMV”, “HRV + HPIV1”, “HRV + HPIV2”, “IVB + EB”, “HMPVA2b + RSV”, and “HMPV1b + HPIV2”.

[0044] For each combination in the above two categories, the detection performance was verified using the corresponding template according to the aforementioned dual detection system and procedure. The results are shown in Table 18. In the 10 combinations with primer-probe heterodimer linking bases <6 bp, no non-specific amplification was observed, and all combinations could detect 5 copies / μL plasmid template. In the 10 combinations with primer-probe heterodimer linking bases ≥6 bp, both specificity and sensitivity did not meet the requirements and could not be used to construct dual systems.

[0045] Target duplications were removed from 10 effective duplex combinations, and 6 isothermal duplex reaction solutions were established: “NCP + HADV3”, “RSV + HPIV2”, “HRV + HADV55”, “IVB + HCMV”, “HMPV1b + HPIV1”, and “HMPVA2b + HPIV3”.

[0046] Table 18 Validation Results of the Dual Isothermal Reaction Liquid Combined Target

[0047] 2. Validation and establishment of triple isothermal detection reaction solution 1) The WNC probes were used to analyze the heterodimers of the six binary combinations ("NCP + HADV3", "IVB + HCMV", "RSV + HPIV2", "HRV + HADV55", "HMPV1b + HPIV1", "HMPVA2b + HPIV3") to determine the number of heterodimer bases linked between the primers and probes for each target. The results are shown in Table 19.

[0048] Table 19 Information on the number of bases linked to the heterodimers formed by the internal reference probe and the target primers / probes in the six binary combinations.

[0049] 2) Enzyme digestion probe isothermal triple detection system and detection procedure (Table 20): Table 20 Components and Detection Procedure of the Triple System for Enzyme Digestion Probe Isothermal Detection

[0050] 3) Experimental verification of the triple system target combination Based on the number of linked bases in the heterodimer, the verification was divided into two categories for comparative verification: Category 1: target combinations with <6 bp linked bases in the primer-probe heterodimer; Category 2: target combinations with ≥6 bp linked bases in the primer-probe heterodimer.

[0051] The first category of target combinations includes: “IVB+HCMV+WNC”, “HRV + HADV55+WNC”, and “HMPVA2b +HPIV3+WNC”.

[0052] The second category of target combinations includes: “NCP + HADV3 + WNC”, “RSV + HPIV2 + WNC”, and “HMPV1b + HPIV1 + WNC”.

[0053] For each combination in the above two categories, the detection performance was verified using the corresponding template according to the aforementioned triple detection system and procedure. The results are shown in Table 21. In the three combinations with primer-probe heterodimer linker bases <6 bp, both the specificity and sensitivity of the amplification met the requirements: no non-specific amplification was observed, and 5 copies / μL plasmid template could be detected. In the three combinations with primer-probe heterodimer linker bases ≥6 bp, neither the specificity nor the sensitivity met the requirements, and they could not be used to construct the triple system.

[0054] Of the three valid combinations, the triplet combinations “IVB+HCMV+WNC” and “HRV + HADV55+WNC” each contained 19 primers and probes, while the combination “HMPVA2b + HPIV3+WNC” contained 18 primers and probes. Considering minimizing the total number of primers and probes, the combination “HMPVA2b + HPIV3+WNC” was ultimately chosen to establish a triplet isothermal reaction solution.

[0055] Table 21 Validation results of the triple isothermal reaction liquid combination target

[0056] 3. Optimization design and validation for heterodimers linking ≥6 bp bases Table 8 shows that the primer-probe heterodimer linkages for EB virus and other viral targets all had bases ≥6 bp. Furthermore, in the experimental verifications described above, the combination effect of this target with other targets was poor. After the above verifications, only IVA and EB remained uncombined among the 14 targets. Therefore, an optimized design and further verification were conducted for the combination of EB virus and IVA.

[0057] 1) EB probe optimization design Table 6 shows that the heterodimer linking bases between the influenza A detection probe and the EB primer probe are all <6 bp. Table 8 shows that the IVA-LF heterodimer linking bases between the EB virus detection probe and the influenza A primer are ≥6 bp. Therefore, the probe sequences screened in the EB singlet were modified by removing the first two bases at the 5' end and changing them to "CGAGACCCGAGTGGCCCGG". ATCC (SEQ ID No. 97) was then compared with the primers for influenza A, and it was found that the number of bases linked by the heterodimer was all <6bp (Table 22).

[0058] Table 22 Comparison results of heterodimers between primers and probes after optimizing primer sequences.

[0059] 2) Performance comparison and verification of EB probe before and after optimization Following the aforementioned isothermal double detection system and procedure, the detection performance of the EB probe before and after optimization was verified using the corresponding template. The results are shown in Table 23. The "EB+ IVA" target combination with optimized EB probes (i.e., primer-probe heterodimer linking <6 bp) achieved the required specificity and sensitivity. However, the combination without optimized primers (i.e., primer-probe heterodimer linking ≥6 bp) could not detect 10 copies / μL of plasmid, and its sensitivity did not meet the requirements, making it unsuitable for constructing double systems.

[0060] Thus, an isothermal double reaction solution, "IVA+EB", was established, further demonstrating that modifying the primer sequence before experimental verification to ensure that the number of bases in the heterodimer linker between the target and primer can be less than 6 bp can effectively improve the success rate of target combination.

[0061] Table 23 Performance validation results of primer sequence optimization in “EB+IVA” dual nucleic acid reaction solution

[0062] It should be understood that the disclosed invention is not limited to the specific methods, schemes, and substances described, as these are all subject to variation. It should also be understood that the terminology used herein is for the purpose of describing specific embodiments only and is not intended to limit the scope of the invention, which is limited only by the appended claims.

[0063] Those skilled in the art will also recognize, or be able to identify, many equivalents of the specific embodiments of the invention described herein using no more than conventional experiments. These equivalents are also included in the appended claims.

Claims

1. A method for multiplex detection by selecting different combinations of target to be detected in an isothermal detection method of enzyme-cleavage probe, wherein specific isothermal amplification primers and fluorescent probes RNHP are designed for each target to be detected, and different fluorescent probes RNHP are labeled with different fluorescent signals for different targets to be detected so that different targets to be detected can be distinguished in different fluorescent signal channels; the fluorescent probe RNHP comprises at least one RNA base, a fluorescent group is labeled on the probe base close to the 5' end of the probe on the left side of the RNA base, and a quenching group is labeled on the probe base close to the 3' end of the probe; the length of the fluorescent probe RNHP is 16-45 bp, the distance between the fluorescent group and the quenching group labeled on the fluorescent probe RNHP is 5-15 bp; the G+C content in the probe fragment on the left side of the RNA base is 40%-60%; the isothermal amplification is LAMP isothermal amplification, and the method is characterized in that, The method comprises respectively designing corresponding enzyme digestion probe isothermal detection probe RNHP and primer for each target to be detected, then comparing the first fluorescent probe RNHP of the first target to be detected with the second fluorescent probe RNHP of the second target to be detected and the LAMP isothermal amplification of all primers of the second target to be detected, if the first fluorescent probe RNHP respectively forms corresponding heterodimer with the second fluorescent probe RNHP of the second target to be detected and the LAMP isothermal amplification of all primers of the second target to be detected, the number of bases of each heterodimer continuously complementary to each other is less than 6, not including 6; at the same time, if the second fluorescent probe RNHP respectively forms corresponding heterodimer with the first fluorescent probe RNHP of the first target to be detected and the LAMP isothermal amplification of all primers of the first target to be detected, the number of bases of each heterodimer continuously complementary to each other is less than 6, not including 6, then the first target to be detected and the second target to be detected can be combined to realize multiplex detection in the enzyme digestion probe isothermal detection method.

2. A multiplex detection kit for different combinations of target to be detected in an isothermal detection method of enzyme-cutting probe, the kit comprising specific isothermal amplification primers and fluorescent probe RNHP designed for each target to be detected, each target to be detected being labeled with different fluorescence by the fluorescent probe RNHP, so that different targets to be detected can be distinguished in different fluorescence signal channels; the fluorescent probe RNHP comprises at least one RNA base, a fluorescent group is labeled on the probe base near the 5' end of the probe on the left side of the RNA base, and a quenching group is labeled on the probe base near the 3' end of the probe; the length of the fluorescent probe RNHP is 16-45 bp, the distance between the fluorescent group and the quenching group labeled on the fluorescent probe RNHP is 5-15 bp; the G+C content in the probe fragment on the left side of the RNA base is 40%-60%; the isothermal amplification is LAMP isothermal amplification, characterized in that, The kit comprises respectively designing corresponding enzyme digestion probe isothermal detection probe RNHP and primer for each target to be detected, comparing the first fluorescent probe RNHP of the first target to be detected with the second fluorescent probe RNHP of the second target to be detected and the LAMP isothermal amplification of all primers of the second target to be detected, the first fluorescent probe RNHP respectively forms corresponding heterodimer with the second fluorescent probe RNHP of the second target to be detected and the LAMP isothermal amplification of all primers of the second target to be detected, the number of bases of each heterodimer continuously complementary to each other is less than 6, not including 6; at the same time, the second fluorescent probe RNHP respectively forms corresponding heterodimer with the first fluorescent probe RNHP of the first target to be detected and the LAMP isothermal amplification of all primers of the first target to be detected, the number of bases of each heterodimer continuously complementary to each other is less than 6, not including 6.

3. The kit for isothermal detection of 14 respiratory infection viruses by enzyme-cleavage probe, characterized in that, The kit effectively combines 7 multiplex reaction solutions for 14 viral targets: reaction solution 1 detects novel coronavirus and adenovirus type 3 / 7; reaction solution 2 detects respiratory syncytial virus and parainfluenza virus type 2; reaction solution 3 detects influenza A virus and EB virus; reaction solution 4 detects rhinovirus A1 and adenovirus 55; reaction solution 5 detects cytomegalovirus and influenza B virus; reaction solution 6 detects metapneumovirus B1 and parainfluenza virus type 1; reaction solution 7 detects metapneumovirus A2b, parainfluenza virus type 3 and internal reference.

4. The kit of claim 3, wherein The LAMP isothermal amplification primers and fluorescent probe RNHP for detecting novel coronavirus in reaction solution 1 are respectively SEQ ID No. 1-SEQ ID No. 6, SEQ ID No. 7, and the LAMP isothermal amplification primers and fluorescent probe RNHP for detecting adenovirus type 3 / 7 are SEQ ID No. 15-SEQ ID No. 19, SEQ ID No.

20.

5. The kit of claim 3, wherein The reaction solution 2 includes LAMP isothermal amplification primer and fluorescent probe RNHP for detecting respiratory syncytial virus as SEQ ID No. 8-SEQ ID No. 13, SEQ ID No. 14, and LAMP isothermal amplification primer and fluorescent probe RNHP for detecting parainfluenza virus type 2 as SEQ ID No. 79-SEQ ID No. 83, SEQ ID No.

84.

6. The kit of claim 3, wherein The reaction solution 3 includes LAMP isothermal amplification primer and fluorescent probe RNHP for detecting influenza virus A as SEQ ID No. 27-SEQ ID No. 33, SEQ ID No. 97, and LAMP isothermal amplification primer and fluorescent probe RNHP for detecting Epstein-Barr virus as SEQ ID No. 41-SEQ ID No. 45, SEQ ID No.

97.

7. The kit of claim 3, wherein The reaction solution 4 includes LAMP isothermal amplification primer and fluorescent probe RNHP for detecting rhinovirus A1 as SEQ ID No. 47-SEQ ID No. 52, SEQ ID No. 53, and LAMP isothermal amplification primer and fluorescent probe RNHP for detecting adenovirus 55 as SEQ ID No. 21-SEQ ID No. 25, SEQ ID No.

26.

8. The kit of claim 3, wherein The reaction solution 5 includes LAMP isothermal amplification primer and fluorescent probe RNHP for detecting cytomegalovirus as SEQ ID No. 54-SEQ ID No. 59, SEQ ID No. 60, and LAMP isothermal amplification primer and fluorescent probe RNHP for detecting adenovirus type 2 as SEQ ID No. 35-SEQ ID No. 39, SEQ ID No.

40.

9. The kit of claim 3, wherein The reaction solution 6 includes LAMP isothermal amplification primer and fluorescent probe RNHP for detecting metapneumovirus B1 as SEQ ID No. 61-SEQ ID No. 64, SEQ ID No. 65, and LAMP isothermal amplification primer and fluorescent probe RNHP for detecting parainfluenza virus type 1 as SEQ ID No. 72-SEQ ID No. 77, SEQ ID No.

78.

10. The kit of claim 3, wherein The reaction solution 7 includes LAMP isothermal amplification primer and fluorescent probe RNHP for detecting metapneumovirus A2b as SEQ ID No. 66-SEQ ID No. 70, SEQ ID No. 71, LAMP isothermal amplification primer and fluorescent probe RNHP for detecting parainfluenza virus type 3 as SEQ ID No. 85-SEQ ID No. 89, SEQ ID No. 90, and LAMP isothermal amplification primer and fluorescent probe RNHP for detecting internal reference as SEQ ID No. 91-SEQ ID No. 95, SEQ ID No. 96.

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