CN117757990A - Respiratory tract adenovirus typing nucleic acid detection primer combination, kit and detection method - Google Patents
Respiratory tract adenovirus typing nucleic acid detection primer combination, kit and detection method Download PDFInfo
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Abstract
The invention discloses a primer combination, a kit and a detection method for detecting respiratory tract adenovirus typing nucleic acid, which are designed aiming at the related adenoviruses of the subgenera B and E of human adenovirus including HAdV-3, HAdV-4, HAdV-7, HAdV-14, HAdV-16, HAdV-21 and HAdV-55 types, and are matched with a proper probe for detection, and meanwhile, a multiplex real-time fluorescent PCR detection kit is established, so that the rapid and accurate identification of a plurality of main types of respiratory tract adenoviruses is realized, the kit can be used for rapid identification of respiratory tract adenovirus typing and epidemiological research of respiratory tract virus infection, and has good practical value.
Description
Technical Field
The invention belongs to the technical field of molecular biology, relates to virus detection, and in particular relates to a respiratory tract adenovirus typing nucleic acid detection primer combination, a kit and a detection method.
Background
Human adenovirus (HAdV) is a DNA virus with high transmission capacity, its structure is non-enveloped, and the viral capsid presents icosahedral symmetry. Since 1953, rowe et al first extracted HadV from atrophic tonsillar adenoids in healthy people, it has been of interest to the infectious and epidemiological community. It has been reported that human adenoviruses now found are classified into seven subgenera A-G, and 113 types based on their own biological structural properties, biological molecules, and genetic and biochemical characteristics. Among the seven different subgenera, pathogenic adenoviruses are most commonly seen as respiratory adenoviruses, wherein three subgenera of B (HAdV-3, 7, 11, 14, 16, 21, 50, 55), C (HAdV-1, 2, 5, 6, 57) and E (HAdV-4) are related to respiratory adenoviruses, and all of the viruses cause respiratory diseases such as upper respiratory tract infection, lower respiratory tract infection and bronchitis, and gastrointestinal symptoms such as diarrhea, vomiting and abdominal pain are accompanied by some cases, and most patients are slightly self-limiting infections, but severe cases also cause pneumonia.
There are a total of 17 types of viruses reported in China that are involved in respiratory tract adenovirus infection, of which HAdV-55, HAdV-7 and HAdV-3 are the major causative genotypes in outbreaks, and in addition, HAdV-4, HAdV-14, HAdV-16, HAdV-21 and the like are also detected in numerous large-scale infections.
The current detection method of the respiratory adenovirus is mainly based on the PCR technology, and has the advantages of sensitivity, rapidness and the like. The multiplex real-time fluorescent PCR technology has the advantages of high specificity and sensitivity, full-closed amplification, simplicity, convenience, rapidness, good repeatability, real-time detection and the like, and becomes an effective means for adenovirus typing diagnosis. However, at present, the respiratory tract adenovirus molecular diagnosis product in China still mainly adopts adenovirus general type detection, but the kit for further typing detection of the adenovirus is fewer, and the related adenovirus typing index is not fully covered. In addition, the HAdV-55 type is a recombinant virus based on a HAdV-14 type genome skeleton chimeric HAdV-11 type Hexon partial fragment, so that a certain cross exists between the HAdV-55 type and the HAdV-14 type, and the existing detection method is difficult to accurately identify the typing.
Therefore, how to rapidly detect and identify multiple major subtypes of respiratory adenoviruses remains a major challenge.
Disclosure of Invention
Aiming at how to rapidly detect and identify a plurality of main types of respiratory adenovirus, the invention provides a respiratory adenovirus typing nucleic acid detection primer combination, a kit and a detection method, wherein primers are designed aiming at the related adenovirus types of the subgenera B and the subgenera E of the human adenovirus, and are matched with a proper probe for detection, and meanwhile, a multiplex real-time fluorescence PCR detection kit is established, so that the rapid and accurate identification of the plurality of main types of the respiratory adenovirus is realized. The specific technical scheme is as follows:
first, the invention provides a respiratory tract adenovirus typing nucleic acid detection primer combination, which comprises eight primer pairs and eight probes, wherein the nucleotide sequences of the primers and the probes are shown as SEQ NO. 1-SEQ NO. 24.
The aforementioned respiratory adenovirus typing nucleic acid detection primer combination, wherein,
the primer pair and the probe shown in SEQ NO. 1-SEQ NO.3 are used for detecting the HAdV-3 type;
the primer pair and the probe shown in SEQ NO. 4-SEQ NO.6 are used for detecting the HAdV-4 type;
the primer pair and the probe shown in SEQ NO. 7-SEQ NO.9 are used for detecting the HAdV-7 type;
the primer pair and the probe shown in SEQ NO. 10-SEQ NO.12 are used for detecting the HAdV-16 type;
the primer pair and the probe shown in SEQ NO. 13-SEQ NO.15 are used for detecting the HAdV-21 type;
the primer pair and the probe shown in SEQ NO. 16-SEQ NO.18 are used for detecting the HAdV-14 type;
the primer pair and the probe shown in SEQ NO. 19-SEQ NO.21 are used for detecting the HAdV-55 type target 1;
the primer pair and the probe shown in SEQ NO. 22-SEQ NO.24 are used for detecting the HAdV-55 type target 2.
The target 1 and the target 2 of the HAdV-55 type are the penton gene and the poly gene respectively.
Secondly, the invention provides a respiratory tract adenovirus typing nucleic acid detection kit, which comprises a reaction mixed solution A, a reaction mixed solution B, a reaction mixed solution C, a hot start DNA polymerase, a HAdV positive quality control product and a negative quality control product;
the reaction mixed solution A, the reaction mixed solution B and the reaction mixed solution C all contain reaction buffer solution and Mg 2+ Deoxynucleotide triphosphate mixtures;
and each reaction mixture also contains one or more groups of primers and probes according to any one of claims 1-3.
In the respiratory tract adenovirus typing nucleic acid detection kit,
the reaction mixture A contains primers and probes for detecting the HAdV-3 type, the HAdV-4 type and the HAdV-7 type;
the reaction mixture B contains primers and probes for detecting the types HAdV-16, HAdV-21 and HAdV-14;
the reaction mixture C contains primers and probes for detecting target 1 and target 2 of type HAdV-55.
The HAdV positive quality control is a recombinant plasmid containing each genotyping detection target sequence of adenovirus; the concentrations are respectively as follows:
HAdV-3：2.42E+06copies/mL，
HAdV-4：1.91E+06copies/mL，
HAdV-7：2.07E+06copies/mL，
HAdV-16：1.88E+06copies/mL，
HAdV-21：1.59E+06copies/mL，
HAdV-14：2.77E+06copies/mL，
HAdV-55：1.38E+06copies/mL；
the negative quality control product is purified water which does not contain RNase and DNase;
the lower limit of detection of the kit for each adenovirus type is 500copies/ml.
The storage condition of the kit is-20 ℃, and the repeated freezing and thawing times are not more than 5 times.
Thirdly, the invention provides a method for detecting the respiratory adenovirus typing nucleic acid, which uses the kit for detection; the PCR reaction system used for detection by the kit is 25. Mu.l, and comprises 20. Mu.l of reaction mixture A or reaction mixture B or reaction mixture C and 5. Mu.l of nucleic acid sample to be detected.
In the aforementioned method for detecting respiratory tract adenovirus genotyping nucleic acid, the components of the reaction mixture A, the reaction mixture B and the reaction mixture C specifically include: 2 XOne Step RT-qPCR Probe Buffer IV 12.5.5 μl; 0.37 to 0.58 mu l of each primer and probe; 0.5. Mu.l of DNA polymerase; the balance of sterilized water.
The reaction conditions of the detection method for the respiratory tract adenovirus typing nucleic acid are as follows: a pre-denaturation stage: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec; a total of 40 cycles; the fluorescent signal is detected during the annealing step.
The invention has the following beneficial effects:
1) The invention combines epidemiological research of respiratory adenovirus, designs specific primers and fluorescent labeling probes aiming at adenovirus typing of the subgenera B and E of human adenovirus, and simultaneously detects a plurality of main typing nucleic acids of respiratory adenovirus, thereby realizing rapid and accurate identification of a plurality of main typing of respiratory adenovirus.
2) The invention develops a multiplex real-time fluorescence PCR detection kit for a plurality of main typing nucleic acids (HAdV-3, HAdV-4, HAdV-7, HAdV-16, HAdV-21, HAdV-14 and HAdV-55) of respiratory adenovirus; the rapid and accurate identification of a plurality of main types of respiratory adenovirus is convenient to realize.
3) Based on the fact that the HAdV-55 type is a recombinant virus of HAdV-14 type genome framework chimeric HAdV-11 type Hexon partial fragment, a certain crossing problem exists between the HAdV-55 type and the HAdV-14 type, a double-target detection mode is created originally aiming at the respiratory tract adenovirus HAdV-55 type, and identification accuracy of the adenovirus HAdV-55 type and the adenovirus HAdV-14 type is improved;
4) The detection method can simultaneously carry out qualitative detection and identification on a plurality of main types of the respiratory adenovirus, and has good practical value.
5) The detection method is more convenient and efficient than a single fluorescent PCR method, and can be used for rapid identification of respiratory adenovirus typing and epidemiological research of respiratory virus infection.
Drawings
FIG. 1 is an amplification plot of the real-time fluorescence PCR detection of adenovirus typing component A (HAdV-3, HAdV-4, HAdV-7) positive standard of the present invention;
FIG. 2 is an amplification plot of the real-time fluorescence PCR detection of adenovirus typing component B (HAdV-16, HAdV-21, HAdV-14) positive standard of the present invention;
FIG. 3 is an amplification curve diagram of the real-time fluorescence PCR detection of adenovirus typing component C (HAdV-55 target 1, HAdV-55 target 2) positive standard;
FIG. 4 is a graph showing the amplification of adenovirus typing type 3 sensitivity verification according to the present invention;
FIG. 5 is a graph showing the amplification of adenovirus type 4 sensitivity test of the invention;
FIG. 6 is a graph showing the amplification of adenovirus type 7 sensitivity test of the invention;
FIG. 7 is a graph showing the amplification of adenovirus typing type 16 sensitivity test of the present invention;
FIG. 8 is a graph of the amplification curve of adenovirus typing type 21 sensitivity verification according to the present invention;
FIG. 9 is a graph showing the amplification of adenovirus type 14 sensitivity test of the invention;
FIG. 10 is a graph of the sensitivity verification amplification of adenovirus type 55 (target 1) according to the invention;
FIG. 11 is a graph showing the sensitivity verification amplification of adenovirus type 55 (target 2) according to the invention.
Detailed Description
The technical solution of the present invention will be clearly and completely described below with reference to the embodiments and the accompanying drawings, and it is obvious that the described embodiments are only preferred embodiments of the present invention, not all embodiments, nor other forms of limitation of the present invention, and any person skilled in the art may make changes or modifications and equivalent variations using the disclosed technical matters. However, any simple modification, equivalent variation and variation of the above embodiments according to the technical substance of the present invention still fall within the protection scope of the technical solution of the present invention.
Example 1
This example provides a respiratory adenovirus typing nucleic acid detection primer combination, a kit and a detection method for rapid detection and identification of a plurality of major typing adenoviruses of the subgenera B and E of human adenoviruses, including the type HAdV-3, the type HAdV-4, the type HAdV-7, the type HAdV-14, the type HAdV-16, the type HAdV-21 and the type HAdV-55. It should be noted that the examples set forth herein are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way. The reagents used therein, such as kits, buffers, etc., are only those specifically selected in this particular example, and it is understood that those skilled in the art can select corresponding reagents of other companies as needed to achieve the object of the present invention.
Wherein, since the HAdV-55 type is a recombinant virus based on a HAdV-14 type genome backbone chimeric HAdV-11 type Hexon partial fragment, a certain cross exists between the HAdV-55 type and the HAdV-14 type. Therefore, aiming at the respiratory tract adenovirus HAdV-55, the embodiment creates a double-target detection mode, selects the penton gene as a target 1, selects the poly gene as a target 2, and respectively designs primers for detection so as to improve the identification accuracy of the adenovirus HAdV-55 and the adenovirus HAdV-14. In this example, the designed upstream primer and downstream primer of each group and the corresponding probe sequences of different fluorescent markers are shown in sequence table 1.
TABLE 1 primer and probe sequences
As shown in Table 1, in this example, the 5' -end of the specific probe sequences of adenovirus HAdV-3, HAdV-16 and HAdV-55 type targets 1 are labeled with FAM fluorophores; the 5' -end of a specific probe sequence of adenovirus HAdV-4 type, HAdV-21 type and HAdV-55 type targets 2 is marked by a VIC fluorescent group; the 5' -end of the adenovirus HAdV-7 specific probe sequence is marked by CY5 fluorescent groups; the 5' -end of the adenovirus HAdV-14 specific probe sequence is marked by ROX fluorescent groups so as to distinguish detection results.
In this embodiment, for the adenovirus typing detection, a multiplex real-time fluorescent PCR detection kit is also designed, which comprises 3 reaction mixtures A, B, C for HAdV typing detection, a hot start DNA polymerase, a positive quality control and a negative quality control of HAdV. The reaction mixture contains reaction buffer solution, a mixture of Mg2+ and deoxynucleotide triphosphate (dNTP), different adenovirus typing gene specific primers, taqMan fluorescent probes and the like. The 3 different primer probes in the reaction mixture are respectively: the reaction mixture A contains HAdV-3 type, HAdV-4 type and HAdV-7 type; the reaction mixture B contains HAdV-16 type, HAdV-21 type and HAdV-14 type; the reaction mixture C contains two gene detection targets of HAdV-55 type. The positive quality control material is a recombinant plasmid (without biological hazard) containing adenovirus each typing detection target sequence; the negative quality control material is water without RNase and DNase.
When the kit is used for detection, a PCR reaction system of 25 mu l is selected, wherein the PCR reaction system comprises 20 mu l of reaction mixed solution A or reaction mixed solution B or reaction mixed solution C and 5 mu l of nucleic acid sample to be detected. The reaction mixed solution A, the reaction mixed solution B and the reaction mixed solution C specifically comprise the following components: 2 XOne Step RT-qPCR Probe Buffer IV 12.5.5 μl; 0.37 to 0.58 mu l of each primer and probe; 0.5. Mu.l of DNA polymerase; the balance of sterilized water.
The specific dosages of the primer and the probe contained in the reaction mixed solution A, the reaction mixed solution B and the reaction mixed solution C are as follows:
the PCR reaction program of the kit is as follows: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec; for a total of 40 cycles. The fluorescent signal is detected during the annealing step.
And (3) quality control: negative and positive controls are set up in each experiment, the negative control has no Ct value (or Ct value is 0), the Ct value of the positive control is less than or equal to 30, otherwise, the experimental result is not established.
Interpretation of the results:
positive: an S-shaped amplification curve appears, and the Ct value is less than or equal to 35; suspicious: an "S" type amplification curve occurs, but Ct values > 35; negative: the "S" type amplification curve does not appear, or the curve is not "S" type although exceeding the threshold; for suspicious results, the experiment should be repeated, and if the repeated experiment or the S-shaped amplification curve appears, the negative control has no pollution, and can be judged to be positive.
The respiratory adenovirus typing detection kit of the embodiment is stored at the temperature of minus 20 ℃ to reduce repeated freeze thawing as much as possible, and the times of freeze thawing are not more than 5 times.
Sample requirements: clinical sample types include pharyngeal swabs, sputum and bronchoalveolar lavage; after clinical samples are collected, the samples are transported with ice and stored at the temperature of minus 20 ℃ and can not be repeatedly frozen and thawed; the method for extracting the nucleic acid from the clinical sample is suggested to adopt a commercial kit with stable and reliable performance, the specific method is referred to the specification of the corresponding commercial kit, the extracted nucleic acid should be detected immediately, or the nucleic acid should be stored at-80 ℃ to-20 ℃ after being split-packed.
The kit is a multiplex real-time fluorescence PCR detection kit, comprises specific primers and fluorescence labeling probes for adenovirus typing of subgenera B and E of human adenovirus, and can be used for simultaneous qualitative detection and identification of a plurality of main typing nucleic acids (HAdV-3, HAdV-4, HAdV-7, HAdV-16, HAdV-21, HAdV-14 and HAdV-55) of respiratory adenovirus; and aiming at the respiratory tract adenovirus HAdV-55, the invention creates a double-target detection mode and improves the identification accuracy of the adenovirus HAdV-55 and the adenovirus HAdV-14. Compared with a single fluorescent PCR method, the method is more convenient and efficient, realizes rapid and accurate identification of a plurality of main types of respiratory adenovirus, and is beneficial to epidemiological research of respiratory virus infection.
Example 2
This example is the construction and validation process of the multiplex real-time fluorescent PCR detection kit described in example 1. The method comprises the following steps:
1. design and synthesis of primers and TaqMan probes
Adenovirus HAdV-3, HAdV-4, HAdV-7, HAdV-16, HAdV-21, HAdV-14 and HAdV-55 gene sequences in Genbank and domestic and foreign documents are downloaded by using NCBI Blast tool, sequences are compared and analyzed by using Bioeditor software, stable conserved regions are respectively selected and determined as detection target sequences, and primers and probes are designed for the sequences of the conserved regions by using Primer expression 3.0 software. The 5 'end fluorescent group of the specific probe can be selected from FAM/VIC (or HEX) ROX/CY5 and other different fluorescent labels, and the 3' end can be correspondingly selected from corresponding quenching groups. Wherein the 5' -end of the specific probe sequence of adenovirus HAdV-3 type, HAdV-16 type and HAdV-55 type targets 1 is marked by FAM fluorescent groups; the 5' -end of a specific probe sequence of adenovirus HAdV-4 type, HAdV-21 type and HAdV-55 type targets 2 is marked by a VIC fluorescent group; the 5' -end of the adenovirus HAdV-7 specific probe sequence is marked by CY5 fluorescent groups; the 5' -end of the adenovirus HAdV-14 specific probe sequence is marked by ROX fluorescent group.
And (3) performing NCBI Blast verification on the designed primer and probe sequences, selecting a design with higher specificity, finally screening out the primer probe sequences shown in the table 1, and synthesizing the primers and probes by a biological engineering (Shanghai) stock company.
2. Preparation for detecting clinical samples and viral nucleic acids
The positive clinical sample in the embodiment is derived from a throat swab sample of a patient with respiratory tract infection and suspected patient of a third-party inspection agency; the positive standard used, namely virus cultures (comprising detection target sequences of type HAdV-3, type HAdV-4, type HAdV-7, type HAdV-14, type HAdV-16, type HAdV-21 and type HAdV-55) were purchased from Pond flourishing organisms in Guangzhou; the negative control substances include nucleic acids such as influenza A virus, influenza B virus, human metapneumovirus, rhinovirus, respiratory syncytial virus, parainfluenza virus, coronavirus, enterovirus, etc.; bacterial nucleic acids such as staphylococcus aureus, pseudomonas aeruginosa, klebsiella pneumoniae and the like are provided by the laboratory. Extracting nucleic acid of each fake product by using nucleic acid extraction or purification reagent produced by Jiangsu and Chuangxiong biotechnology limited company for later use; specific extraction steps are referred to the kit operating instructions.
3. Optimization of reaction conditions
In this example, 20. Mu.l of the reaction system and 25. Mu.l of the reaction system were compared, and the final optimization was performed to determine the template loading of 5. Mu.l using 25. Mu.l of the reaction system, i.e., 20. Mu.l of the adenovirus typing reaction mixture.
Meanwhile, the method is optimized according to the length of the amplified fragment, the annealing temperature of the primer and the probe and the enzyme characteristics, and the annealing temperature and the reaction time of the reaction are mainly optimized, so that a rapid amplification program is determined. The final determined cycle parameters are the pre-denaturation stage: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec; for a total of 40 cycles. The fluorescent signal is detected during the annealing step. After amplification, data analysis is carried out according to the same condition, a typical S-shaped amplification curve is detected by the sample, and Ct value of each sample is determined.
4. Evaluation of detection Limit
In this example, the detection limit of the kit described in example 1 was evaluated using the above positive standard, and the positive standard concentrations were respectively: HAdV-3:2.42E+06copies/mL, HAdV-4:1.91E+06copies/mL, HAdV-7:2.07E+06copies/mL, HAdV-16:1.88E+06copies/mL, HAdV-21:1.59E+06copies/mL, HAdV-14:2.77E+06copies/mL, HAdV-55:1.38E+06copies/mL. Finally, the lower limit of detection of each adenovirus type by the kit described in example 1 was determined to be 500copies/mL.
5. Evaluation of detection specificity
As shown in fig. 1 to 11, in this example, the specificity of the kit described in example 1 was evaluated using the above adenovirus typing positive standard nucleic acid as a template, and a clear amplification curve was observed when each adenovirus typing was detected. When 8 common respiratory viruses in the negative control are used for nucleic acid detection, a positive amplification curve is not generated, which indicates that the cross reaction between the probes and the primers used in the kit described in the example 1 and other selected strains does not exist.
Although certain embodiments of the present invention have been described above by way of example in a preferred manner, it will be understood by those skilled in the art that the present invention is not limited to the embodiments disclosed above, but may be modified in light of the knowledge of those skilled in the art to which the present invention pertains without exceeding the scope of the claimed invention. For example, the fluorescent real-time PCR used in the present invention may also employ, as required, a labeling substance other than the fluorescent reporter group and the fluorescent quencher group indicated in examples listed in the specification, such as a label of Tet, TAMRA, ROX, cy3, txRd, JOE or the like; or other labeling systems than Taqman technology, such as fluorescent probe labeling technologies, e.g., molecular beacon MB probes, scorpion probes, fluorescent double hybridization probes, etc.; or using dye-embedding method such as SYBR Green I and the like unsaturated dye and LC Green and the like saturated dye, and only using the specific primer sequence and the primer ratio, the existence of the target gene can be qualitatively or quantitatively detected, so that the existence of the novel coronaries sgRNA-E and ORF1ab genes can be rapidly and specifically detected by the same reaction system. Therefore, such modifications and alternatives as would be apparent to one skilled in the art are intended to fall within the scope of the present invention. The scope of the invention should be defined by the appended claims.
Claims (10)
1. A respiratory adenovirus typing nucleic acid detection primer combination, which is characterized in that: the kit comprises eight primer pairs and eight probes, wherein the nucleotide sequences of the primers and the probes are shown in SEQ NO. 1-SEQ NO. 24.
2. The respiratory adenovirus typing nucleic acid detection primer combination according to claim 1, wherein: wherein,
the primer pair and the probe shown in SEQ NO. 1-SEQ NO.3 are used for detecting the HAdV-3 type;
the primer pair and the probe shown in SEQ NO. 4-SEQ NO.6 are used for detecting the HAdV-4 type;
the primer pair and the probe shown in SEQ NO. 7-SEQ NO.9 are used for detecting the HAdV-7 type;
the primer pair and the probe shown in SEQ NO. 10-SEQ NO.12 are used for detecting the HAdV-16 type;
the primer pair and the probe shown in SEQ NO. 13-SEQ NO.15 are used for detecting the HAdV-21 type;
the primer pair and the probe shown in SEQ NO. 16-SEQ NO.18 are used for detecting the HAdV-14 type;
the primer pair and the probe shown in SEQ NO. 19-SEQ NO.21 are used for detecting the HAdV-55 type target 1;
the primer pair and the probe shown in SEQ NO. 22-SEQ NO.24 are used for detecting the HAdV-55 type target 2.
3. The respiratory adenovirus typing nucleic acid detection primer combination according to claim 2, wherein: the HAdV-55 type target 1 and target 2 are the penton gene and the poly gene respectively.
4. A respiratory tract adenovirus typing nucleic acid detection kit is characterized in that: comprises a reaction mixed solution A, a reaction mixed solution B, a reaction mixed solution C, a hot start DNA polymerase, a HAdV positive quality control product and a negative quality control product;
the reaction mixed solution A, the reaction mixed solution B and the reaction mixed solution C all contain reaction buffer solution and Mg 2+ Deoxynucleotide triphosphate mixtures;
and each reaction mixture also contains one or more groups of primers and probes according to any one of claims 1-3.
5. The kit for detecting the genotyping nucleic acid of respiratory adenovirus according to claim 4, wherein:
the reaction mixture A contains primers and probes for detecting the HAdV-3 type, the HAdV-4 type and the HAdV-7 type;
the reaction mixture B contains primers and probes for detecting the types HAdV-16, HAdV-21 and HAdV-14;
the reaction mixture C contains primers and probes for detecting target 1 and target 2 of type HAdV-55.
6. The kit for detecting the genotyping nucleic acid of respiratory adenovirus according to claim 4, wherein: the HAdV positive quality control is a recombinant plasmid containing adenovirus each typing detection target sequence; the concentrations are respectively as follows:
HAdV-3：2.42E+06copies/mL，
HAdV-4：1.91E+06copies/mL，
HAdV-7：2.07E+06copies/mL，
HAdV-16：1.88E+06copies/mL，
HAdV-21：1.59E+06copies/mL，
HAdV-14：2.77E+06copies/mL，
HAdV-55：1.38E+06copies/mL；
the negative quality control product is purified water which does not contain RNase and DNase;
the lower limit of detection of the kit for each adenovirus type is 500copies/ml.
7. The kit for detecting the genotyping nucleic acid of respiratory adenovirus according to claim 4, wherein: the storage condition of the kit is-20 ℃, and the repeated freezing and thawing times are not more than 5 times.
8. A method for detecting respiratory tract adenovirus typing nucleic acid is characterized in that: performing a test using the kit of any one of claims 4-7; the PCR reaction system for detection and selection by using the kit is 25 mu l, and comprises 20 mu l of reaction mixed liquid A or reaction mixed liquid B or reaction mixed liquid C and 5 mu l of nucleic acid samples to be detected.
9. The method for detecting a respiratory adenovirus typing nucleic acid according to claim 8, wherein: the reaction mixed solution A, the reaction mixed solution B and the reaction mixed solution C specifically comprise the following components:
2×One Step RT-qPCR Probe Buffer IV 12.5µl；
0.37-0.58 mu l of each primer and probe;
DNA polymerase 0.5 μl;
the balance of sterilized water.
10. The method for detecting a respiratory adenovirus typing nucleic acid according to claim 8, wherein: the reaction conditions for this detection were: a pre-denaturation stage: a pre-denaturation stage: 94 ℃ for 2min; amplification stage: denaturation at 94 ℃,10sec; annealing at 56 ℃ for 50sec for 40 cycles; the fluorescent signal is detected during the annealing step.
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