CN113151602A - Primer probe set for detecting adenovirus type 55, kit and application - Google Patents
Primer probe set for detecting adenovirus type 55, kit and application Download PDFInfo
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Abstract
The invention belongs to the technical field of chemical synthesis and diagnostic reagent detection, and particularly relates to a primer probe set for detecting adenovirus type 55, a kit and application. The primer probe set for detecting adenovirus type 55 comprises a Hexon primer probe set and a Fiber primer probe set; nucleotide sequences of primers and probes in the Hexon primer probe set are shown as SEQIDNO.1-3, and nucleotide sequences of primers and probes in the Fiber primer probe set are shown as SEQIDNO.4-6. The lower limit of the detection sensitivity of the probe and the primer provided by the invention to adenovirus 55 type virus nucleic acid is lower than 46 copies, and the sensitivity is extremely high; when the probe and the primer provided by the invention are used for detecting adenovirus type 1, type 3, type 4, type 5, type 6, type 7 and different rubella viruses of 3 strains, ROX or FAM signals are not found, and the specificity is very high.
Description
Technical Field
The invention belongs to the technical field of chemical synthesis and diagnostic reagents, and particularly relates to a primer probe set for detecting adenovirus type 55, a kit and application.
Background
The capsid of the adenovirus is composed of hexon, penton and fiber proteins, which contain type, group and subgroup epitopes and are the standard for diagnosis of different serotypes of adenovirus.
The existing detection method aiming at the adenovirus is mainly an immunofluorescence-based detection method clinically established in the sixties, but mainly aims at the 3-type and 7-type adenoviruses, and is difficult to accurately detect the adenoviruses of other serotypes in a typing manner.
Disclosure of Invention
In order to solve the problems, the invention provides a primer probe set for detecting adenovirus type 55, a kit and application. The primer probe set provided by the invention can effectively improve the specificity of detection, and when ROX signals and FAM signals are double positive, the detection result can directly point to adenovirus type 55 positive, so that the defect of misjudgment of adenovirus type is effectively avoided.
In order to achieve the purpose, the invention provides the following technical scheme:
the invention provides a primer probe group for detecting adenovirus type 55, which comprises a Hexon primer probe group and a Fiber primer probe group;
the Hexon primer Probe set comprises a forward primer HAdV11-Hexon-F with the nucleotide sequence shown as SEQ ID NO.1, a reverse primer HAdV11-Hexon-R with the nucleotide sequence shown as SEQ ID NO.2 and a Probe HAdV11-Hexon-Probe with the nucleotide sequence shown as SEQ ID NO.3, wherein the 3 'end of the Probe HAdV11-Hexon-Probe is provided with a quenching group, and the 5' end of the Probe HAdV11-Hexon-Probe is provided with a fluorescent group;
the Fiber primer Probe set comprises a forward primer HAdV14-Fiber-F with a nucleotide sequence shown as SEQ ID NO.4, a reverse primer HAdV14-Fiber-R with a nucleotide sequence shown as SEQ ID NO.5 and a Probe HAdV14-Fiber-Probe with a nucleotide sequence shown as SEQ ID NO.6, wherein the 3 'end of the Probe HAdV14-Fiber-Probe is provided with a quenching group, and the 5' end of the Probe HAdV14-Fiber-Probe is provided with a fluorescent group.
Preferably, the quencher group comprises BHQ1 and BHQ2, and the fluorescent group comprises ROX and FAM.
The invention provides application of the primer probe set in detecting adenovirus type 55.
The invention also provides a kit for detecting adenovirus type 55, which comprises the primer probe set and reaction liquid.
Preferably, the reaction solution comprises 2 × PCR Buffer and EX TAQ HS.
The invention provides a primer probe group for detecting adenovirus type 55, which comprises a Hexon primer probe group and a Fiber primer probe group;
the nucleotide sequence of the Hexon primer probe group is shown as SEQ ID NO. 1-3, and the nucleotide sequence of the Fiber primer probe group is shown as SEQ ID NO. 4-6. The primer probe group for real-time fluorescent quantitative detection of adenovirus type 55 provided by the invention analyzes the gene sequences of Hexon and Fiber proteins in adenovirus type 55 by a bioinformatics method, designs and synthesizes a probe with a quenching group and a fluorescent group, and synthesizes an upstream primer and a downstream primer according to the probe. Two groups of primers and probes aiming at the genes of the Hexon and Fiber proteins are synthesized by a chemical synthesis technology, can be used for identifying the HAdV-55 type virus with high specificity, and can also give consideration to the qualitative and quantitative detection of the HAdV-55 type virus. As can be seen from the example data, the lower limit of the detection sensitivity of the probe and the primer provided by the invention to adenovirus type 55 virus nucleic acid is lower than 46 copies, which shows that the probe and the primer have extremely high sensitivity; the probes and primers provided by the invention are used for detecting common respiratory adenovirus 1, 3, 4, 5, 6, 7 and 3 different rubella viruses, and no ROX or FAM signal is found, which indicates that the probe and the primers have extremely high specificity.
Drawings
FIG. 1 is a diagram showing the results of detection of adenovirus type 55 nucleic acid;
FIG. 2 is a diagram showing the results of detection of adenovirus type 55 nucleic acid;
FIG. 3 is a graph of the nucleotide copy number of adenovirus type 55 versus the average of CT values for ROX signal;
FIG. 4 is a graph of nucleotide copy number of adenovirus type 55 versus the mean number of CT values for FAM signal;
FIG. 5 is a diagram showing the results of detection of adenovirus type 3, 4 and 7 nucleic acids;
FIG. 6 is a diagram showing the results of nucleic acid detection of adenovirus type 1, 3, 5, 6 and 3 rubella viruses.
Detailed Description
The invention provides a primer probe group for detecting adenovirus type 55, which comprises a Hexon primer probe group and a Fiber primer probe group;
the Hexon primer Probe set comprises a forward primer HAdV11-Hexon-F with the nucleotide sequence shown as SEQ ID NO.1, a reverse primer HAdV11-Hexon-R with the nucleotide sequence shown as SEQ ID NO.2 and a Probe HAdV11-Hexon-Probe with the nucleotide sequence shown as SEQ ID NO.3, wherein the 3 'end of the Probe HAdV11-Hexon-Probe is provided with a quenching group, and the 5' end of the Probe HAdV11-Hexon-Probe is provided with a fluorescent group;
the SEQ ID NO.1 is TCAGTGGATTGCAGAAGGTG, the SEQ ID NO.2 is TCAGCTTTTACGGGAGCATT, and the SEQ ID NO.3 is AGGAGCGCGTAACAGAAGAGGAAAACAA;
in the invention, the quenching group comprises BHQ1 and BHQ2, the fluorescent group comprises ROX and FAM, in the practical application of the invention, the quenching group of the Probe HAdV11-hexon-Probe is preferably BHQ1, and the fluorescent group is preferably FAM;
the Fiber primer Probe set comprises a forward primer HAdV14-Fiber-F with a nucleotide sequence shown as SEQ ID NO.4, a reverse primer HAdV14-Fiber-R with a nucleotide sequence shown as SEQ ID NO.5 and a Probe HAdV14-Fiber-Probe with a nucleotide sequence shown as SEQ ID NO.6, wherein the 3 'end of the Probe HAdV14-Fiber-Probe is provided with a quenching group, and the 5' end is provided with a fluorescent group;
the SEQ ID NO.4 is TGACACTGATGGGACCTTAC, the SEQ ID NO.5 is TTTTCATCTGTTCCCAATCC, and the SEQ ID NO.6 is CCACACCACTTGTTAAGACTGGGCA;
in the invention, the quenching group comprises BHQ1 and BHQ2, the fluorescent group comprises ROX and FAM, in the practical application of the invention, the quenching group of the Probe HAdV14-Fiber-Probe is preferably BHQ2, and the fluorescent group is preferably ROX;
the primer probe set provided by the invention has high sensitivity and high specificity when detecting the HAdV-55 type virus, and can enable the detection result to directly point to adenovirus 55 type positive when ROX signals and FAM signals are double positive.
The invention provides application of the primer probe set in detecting adenovirus type 55. In the invention, the application can be used for carrying out real-time fluorescence quantitative detection on the HAdV-55 type virus, and can effectively avoid the situation of wrong judgment of the adenovirus type.
The invention also provides a kit for detecting adenovirus type 55, which comprises the primer probe set and reaction liquid; the reaction solution preferably includes 2 XPCR Buffer and EX TAQ HS. The kit provided by the invention has high sensitivity, and the lower limit of the detection sensitivity is lower than 46 copies; the specificity is strong, and adenovirus type 55 can be accurately detected from various adenoviruses and other types of viruses.
In the present invention, the detection method of the kit is preferably qPCR method; and when the ROX signal and the FAM signal are detected to be double positive by the qPCR method, the type 55 adenovirus is positive.
The reaction system of the qPCR method preferably comprises the following:
the reaction conditions of the qPCR method are preferably: 2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 40 cycles.
For further illustration of the present invention, the primer probe set, the kit and the application for detecting adenovirus type 55 provided by the present invention are described in detail below with reference to the drawings and examples, but they should not be construed as limiting the scope of the present invention.
Examples
Firstly, extracting nucleic acid
The sample nucleic acid extraction work was performed in biosafety P2 laboratory.
The instrument comprises the following steps: an automatic nucleic acid extractor manufactured by the company Sainan-Long, Inc. GeneRotex 96.
The kit comprises: the nucleic acid extraction kit matched with the GeneRotex96 automatic nucleic acid extractor specifically operates as follows:
1) preparing a nucleic acid extraction kit, slowly reversing for several times to resuspend the magnetic beads, and lightly throwing the pore plate to enable the reagent and the magnetic beads to be settled at the bottom of the pore plate;
2) shaking the sample for 10s, and then performing instantaneous centrifugation;
3) respectively adding 200 mu L of samples into the 1 st column and the 7 th column of a nucleic acid extraction kit pore plate;
4) inserting stirring rods into the 2 nd and 8 th rows, and placing the pore plate at the corresponding position of the automatic nucleic acid extractor;
5) after the automatic procedures of cracking, washing, elution, magnetism abandoning and the like are carried out according to the preset procedures, the eluent in the 6 th column and the eluent in the 12 th column are transferred into a sterile EP tube, and the serial number and the time are marked.
Identification of adenovirus type 55 by qPCR method
1) The kit comprises: TakaraPremix Ex Taq (Probe qPCR) (CatNo: RR390A)
2) Primers and probes:
hexon primer probe set
HAdV11-hexon-F：5'-TCAGTGGATTGCAGAAGGTG-3'(SEQ IDNO.1)；
HAdV11-hexon-R：5'-TCAGCTTTTACGGGAGCATT-3'(SEQ IDNO.2)；
HAdV 11-hexon-Probe: 5'-AGGAGCGCGTAACAGAAGAGGAAAACAA-3' (SEQ ID NO. 3); the quenching group is BHQ1 and is connected to the 3 'end, and the fluorescent group is FAM and is connected to the 5' end;
fiber primer probe set
HAdV14-Fiber-F：5'-TGACACTGATGGGACCTTAC-3'(SEQ IDNO.4)；
HAdV14-Fiber-R：5'-TTTTCATCTGTTCCCAATCC-3'(SEQ IDNO.5)；
HAdV 14-Fiber-Probe: 5'-CCACACCACTTGTTAAGACTGGGCA-3' (SEQ ID NO.6), the quenching group is BHQ2, which is connected at the 3 'end, and the fluorescent group is ROX, which is connected at the 5' end.
3) Preparation of reaction solution system
TABLE 1 reaction System
4) Reaction conditions
2min at 95 ℃; 95 ℃ for 5s, 60 ℃ for 30s, 40 cycles.
Third, test results
1. Sensitivity verification
The purified nucleic acid copy number (4.583X 10) was selected and determined9) The adenovirus type 55 virus nucleic acid extract is used as a stock solution to be detected. The stock solution is diluted by 10 times in series, and the highest dilution time is 109And (4) doubling. The adenovirus type 55 virus nucleic acid solutions of 10 concentrations, including the stock solution, were sampled and tested according to the protocol described above, with triplicate wells for each concentration. The test results are shown in table 2, table 3 and fig. 1 to 4.
TABLE 2 sensitivity test (detection of ROX signal)
Note: "/" indicates no detectable signal, as shown in the table below.
TABLE 3 sensitivity test (detection of FAM signals)
As can be seen from Table 2, Table 3 and FIGS. 1 to 4, the results of the tests showed that the stock solution to be tested was diluted 108The ROX and FAM signals of the target of the invention can be detected at double time, and both CT values are less than 38, namely, the lower limit of the detection sensitivity of the invention to adenovirus type 55 virus nucleic acid is less than 46 copies. The primer probe set provided by the invention is extremely high in sensitivity.
2. Specificity verification
And selecting nucleic acid extracting solutions of adenovirus types 1, 3, 4, 5, 6, 7 and 3 rubella viruses as samples to be detected. Each sample to be tested was sampled and tested according to the above procedure, with three wells repeated for each sample to be tested. The test results are shown in table 4, table 5 and fig. 5 and 6.
TABLE 4 ROX signals for other types of adenovirus and rubella virus
TABLE 5 FAM signals of other types of adenovirus and rubella virus
As can be seen from tables 4 and 5 and FIGS. 5 and 6, the test results show that the primer probe set provided by the present invention cannot generate effective ROX or FAM signals in qPCR using the nucleic acid extract of adenovirus type 1, 3, 4, 5, 6, 7 and 3 rubella viruses as the test sample. As can be seen from the detection in tables 2 and 3, the primer probe set provided by the invention can accurately distinguish adenovirus type 55 from various adenovirus types and other types of viruses, which indicates that the primer probe set provided by the invention can accurately point to adenovirus type 55.
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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aggagcgcgt aacagaagag gaaaacaa 28
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Claims (5)
1. The primer probe group for detecting adenovirus type 55 is characterized by comprising a Hexon primer probe group and a Fiber primer probe group;
the Hexon primer Probe set comprises a forward primer HAdV11-Hexon-F with the nucleotide sequence shown as SEQ ID NO.1, a reverse primer HAdV11-Hexon-R with the nucleotide sequence shown as SEQ ID NO.2 and a Probe HAdV11-Hexon-Probe with the nucleotide sequence shown as SEQ ID NO.3, wherein the 3 'end of the Probe HAdV11-Hexon-Probe is provided with a quenching group, and the 5' end of the Probe HAdV11-Hexon-Probe is provided with a fluorescent group;
the Fiber primer Probe set comprises a forward primer HAdV14-Fiber-F with a nucleotide sequence shown as SEQ ID NO.4, a reverse primer HAdV14-Fiber-R with a nucleotide sequence shown as SEQ ID NO.5 and a Probe HAdV14-Fiber-Probe with a nucleotide sequence shown as SEQ ID NO.6, wherein the 3 'end of the Probe HAdV14-Fiber-Probe is provided with a quenching group, and the 5' end of the Probe HAdV14-Fiber-Probe is provided with a fluorescent group.
2. The primer probe set of claim 1, wherein the quencher group comprises BHQ1 and BHQ2, and the fluorophore group comprises ROX and FAM.
3. Use of a primer probe set according to claim 1 or 2 for detecting adenovirus type 55.
4. A kit for detecting adenovirus type 55, comprising the primer probe set of claim 1 or 2 and a reaction solution.
5. The kit according to claim 4, wherein the reaction solution comprises 2 XPCR buffer and EX TAQ HS.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107043831A (en) * | 2017-06-13 | 2017-08-15 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit |
| CN109402067A (en) * | 2018-10-18 | 2019-03-01 | 北京晨寰生物科技有限公司 | One plant of 55 type adenovirus and its vaccine product of preparation |
| CN110964858A (en) * | 2019-12-30 | 2020-04-07 | 瑞普（保定）生物药业有限公司 | Kit for PCR detection and typing of avian adenovirus serum 4 and detection method thereof |
| CN112142830A (en) * | 2020-09-18 | 2020-12-29 | 长江大学 | Recombinant avian adenovirus type 4fiber2 protein and preparation method and application thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107043831A (en) * | 2017-06-13 | 2017-08-15 | 福建省农业科学院畜牧兽医研究所 | Ana 1 aviadenovirus A types and 2 type Real time PCR detection primers, probe and kit |
| CN109402067A (en) * | 2018-10-18 | 2019-03-01 | 北京晨寰生物科技有限公司 | One plant of 55 type adenovirus and its vaccine product of preparation |
| CN110964858A (en) * | 2019-12-30 | 2020-04-07 | 瑞普（保定）生物药业有限公司 | Kit for PCR detection and typing of avian adenovirus serum 4 and detection method thereof |
| CN112142830A (en) * | 2020-09-18 | 2020-12-29 | 长江大学 | Recombinant avian adenovirus type 4fiber2 protein and preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GUO L等: "Detection of three human adenovirus species in adults with acute respiratory infection in China", 《EUR J CLIN MICROBIOL INFECT DIS》 * |
| 刘琳等: "血清4型禽腺病毒TaqMan实时荧光定量PCR检测方法的建立", 《中国兽医学报》 * |
| 朱小娟等: "腺病毒55型感染肺炎患者的病原学鉴定及全基因序列分析", 《中国病原生物学杂志》 * |
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