CN113564280A - RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof - Google Patents
RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof Download PDFInfo
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Abstract
The invention discloses an RAA primer for detecting 12 serotypes of avian adenovirus group I and a detection method thereof, belonging to the technical field of avian virus detection. The sequences of the primers and the probes in the RAA primer and probe combination for detecting 12 serotypes of the avian adenovirus group I are respectively shown as SEQ ID NO.1-SEQ ID NO. 3. The invention can detect FadV of 12 different serotypes simultaneously; in addition, the method only needs necessary reagents and isothermal amplification equipment, is simple and convenient to operate, and is particularly suitable for large-scale detection of the chicken flocks in the basic farm, so that the method is favorable for purifying the FadV of the chicken flocks.
Description
Technical Field
The invention relates to the technical field of avian virus detection, in particular to an RAA primer for detecting 12 serotypes of avian adenovirus group I and a detection method thereof.
Background
Avian adenovirus (fowladenvirus, FAdV) is a DNA virus belonging to the family adenoviridae, the genus adenovirus. FAdV is divided into 3 groups according to antigenicity difference, wherein the FAdV in group I is most pathogenic, and the economic loss is the largest. Group I FAdV was subdivided into 5 subgroups (FAdV A-FAdV E), 12 serotypes (FAdV-1 to 7, FAdV-8a, FAdV-8b, FAdV-9 to 11). The diseases of the chicken caused by the group I FAdV mainly comprise Inclusion Body Hepatitis (IBH), hydropericardium syndrome (HPS), muscular stomach erosion (GE) and the like. Epidemiological investigation shows that in recent years, the FAdV epidemic presents the characteristics of mixed infection of a plurality of serotypes, wherein, FAdV 4 serotype 4 (FAdV-4), FAdV 8a serotype 8a (FAdV-8a) and FAdV 8b serotype 8b (FAdV-8b) are the three serotypes which have the most serious influence on poultry industry in China.
Currently, there are the following main methods for the diagnosis of group I avian adenovirus: firstly, adopting chick embryo or cell to separate pathogeny. Tissues and excrement with high virus content, such as diseased liver, kidney, bursa of fabricius, excrement and the like, can be collected as disease materials, infected disease material tissues are prepared into suspension to be inoculated into SPF chicken embryos, a few of the chicken embryos start to develop and stop within 48 hours after inoculation, the embryo bodies bleed and die, most of the chicken embryos die within 3-6 days, and part of the chicken embryos die after 7 days; cell culture as another culture mode, mainly inoculating the avian adenovirus to chicken liver cancer cells or primary kidney cells for separation and identification. And secondly, adopting an agar gel precipitation test. The test is widely applied in veterinary clinic and is suitable for antibody detection of infected FAdV chicken flocks. Firstly, serum of sick chicken is separated, and is cultured in an incubator at 37 ℃ for more than 24 hours, so that the antigen and the antibody are specifically combined to form an antigen-antibody compound. The results were judged based on whether or not a white precipitate line was observed. And thirdly, enzyme-linked immunosorbent assay, which can detect antigen or antibody for accurate determination, and the prior ELISA is used for detecting the avian adenovirus. In addition, Polymerase Chain Reaction (PCR) technology and real-time fluorescent quantitative PCR technology are also widely used for the etiological detection of FAdV. Generally, the liver of a sick bird is taken, a PCR instrument is used for carrying out PCR reaction after a viral genome is extracted, a large number of target gene fragments are amplified, and FAdV detection is carried out. At present, a plurality of FAdV-directed PCR methods (Yuanyuan, 2016) are established, and after amplification, enzyme digestion can be carried out through a restriction enzyme technology, so that the purpose of distinguishing reference strains of 12 FAdV I serotypes is achieved. In summary, conventional detection methods such as ELISA and agar diffusion assay have low cost and simple operation, but have the disadvantages of long time consumption, poor sensitivity and lower sensitivity than the neutralization assay. The PCR and fluorescent quantitative PCR technology has higher sensitivity, but has higher requirements on test instruments and operators, and is not suitable for large-scale detection in clinical fields.
The Recombinase-mediated Amplification-Lateral flow chromatography (RAA-LFD) technology is characterized in that on the basis of the Recombinase-mediated Amplification technology, the 5 'end of a downstream primer of a reaction is marked by biotin (biotin), an nfo probe with the length of 46 bp-52 bp is added into a reaction system, the 5' end of the probe is marked by 6-carboxyfluorescein (FAM), the position more than 30bp away from the 5 'end is marked by Tetrahydrofuran (THF) residues, the 3' end is phosphorylated and blocked, and the distance from the tetrahydrofuran THF residues is more than 15 bp. After the reaction, a double-labeled product is formed, and the on-site rapid detection is realized by using a Lateral Flow Dipstick (LFD) coated with biotin and FAM antibodies. The RAA-LFD has higher sensitivity, can realize result visualization, basically has no additional purchase of any other auxiliary equipment, only needs a constant temperature device of 39 ℃, can complete reaction by adopting a vacuum cup under extreme conditions to obtain a test result, is suitable for rapid field screening of pathogens, is particularly suitable for the field where experimental equipment is simple and crude and emergency situations, and is widely applied to the field of detection of various pathogens at present.
Patent CN112746135A discloses a primer probe combination for detecting I group 4 avian adenovirus based on RAA technology; patent CN107385110A discloses an RPA primer for detecting avian adenovirus serotype 4; the patent is based on that the RAA technology has better sensitivity and specificity in the detection of the avian adenovirus group I4, but only can detect a single serotype, and cannot realize the simultaneous detection of 12 serotypes of the avian adenovirus group I.
Patent CN106868212A discloses a general type PCR detection primer for avian adenovirus subgroup I, but the general type PCR detection primer only carries out general verification on 4 serotypes and 6 serotypes of avian adenovirus subgroup I, namely A serotype, C serotype, D serotype and E serotype, and the simultaneous detection on 12 serotypes of avian adenovirus subgroup I is not realized.
Disclosure of Invention
In view of the prior art, the invention aims to provide an RAA primer for detecting 12 serotypes of avian adenovirus group I and a detection method thereof.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the invention, there is provided a RAA primer and probe combination for detecting 12 serotypes of avian adenovirus group I, wherein the sequences of the primers and probes in the RAA primer and probe combination are shown in SEQ ID nos. 1 to 3, specifically as follows:
an upstream primer: 5 '-AGGTCCTGTTCGAAGAGGATDCGCGTGGTR-3'; (SEQ ID NO.1)
A downstream primer: 5 '- [ Biotin ] GAHTACGTCAGCAAAAACATCGCVGCCAA-3'; (SEQ ID NO.2)
And (3) probe: TGTTGGTGGCGAAGGCGCCGTAGAGGGCGT [ FAM-dT ] "THF" [ BHQ1-dT ] GAGCATTTGGAGATGG-C3 spacer; (SEQ ID NO.3)
Description of the drawings: in the primer and the probe, D is A/G/T, R is A/G, H is A/C/T, and V is A/C/G.
In a second aspect of the invention, the RAA primer and the probe combination are used for preparing a reagent or a kit for detecting 12 serotypes of avian adenovirus group I.
In a third aspect of the invention, there is provided a kit for detecting 12 serotypes of avian adenovirus group I, the kit comprising the RAA primer and probe combination described above.
Further, the kit further comprises: RAA reagent and lateral flow chromatography test strip.
Preferably, the RAA-reactive reagent comprises: a buffer and B buffer.
In a fourth aspect of the present invention, there is provided a method for detecting 12 serotypes of avian adenovirus group I by using the above kit, comprising the following steps:
and extracting DNA of a sample to be detected as a template, carrying out RAA amplification by using an RAA primer and probe combination in the kit, and detecting an RAA amplification product by using a lateral flow chromatography test strip.
Preferably, the reaction system for RAA amplification is: a buffer 40.9 μ L; b, 2.5 mu L of buffer; 2 mu L of upstream primer; 2 mu L of downstream primer; probe 0.6 μ L; template 2. mu.L.
Preferably, the conditions for RAA amplification are: reacting for 8-15min at the constant temperature of 39 ℃.
Preferably, the method for detecting the RAA amplification product by using the lateral flow chromatography test strip comprises: the RAA-LFD amplification product was added dropwise to the sample pad of the lateral flow chromatography strip, and then the lateral flow chromatography strip was inserted into an EP tube containing 200. mu.L of buffer solution, and the results were observed after 3 min.
The invention has the beneficial effects that:
the invention designs primers and probes aiming at 12 different serological FAdV and establishes a FAdV detection method of RAA-LFD. The detection sensitivity of the method can reach 100 copies/mu l, the repeatability of the test is good, the accuracy is high, and the detection efficiency is greatly improved. And, because the target site designed by the method is located in the FAdV genomes of 12 different serotypes, the method can simultaneously detect the FAdV of 12 different serotypes. In addition, the method has low requirements on equipment, and can be applied in a laboratory and operated on site; the method can be used for detecting the infection of the chicken flock FAdV and detecting the FAdV pollution condition in the poultry attenuated vaccine.
Drawings
FIG. 1: the invention aims at the detection of the sensitivity of the 12 serotypes RAA-LFD method of FadV; in the figure, A-L are positive standard FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10, FAdV-11RAA-LFD detection method sensitivities, respectively; 1 is 10 of each positive standard2copies/. mu.L; and 2 is a negative control.
FIG. 2: PCR method sensitivity detection for 12 serotypes of FAdV; in the figure, A-L are the sensitivity of the normal PCR methods for positive standards FAdV-1, FAdV-2, FAdV-3, FAdV-4, FAdV-5, FAdV-6, FAdV-7, FAdV-8a, FAdV-8b, FAdV-9, FAdV-10 and FAdV-11, respectively; m: 2000DNA Marker; 1 to 7 are each 106、105、104、103、102、101、100copies/. mu.L positive standard; and 8 is a negative control.
FIG. 3: the specificity detection result of the invention; in the figure, 1: FAdV; 2: ALV-A; ALV-J; 4, CIAV; 5, MDV; and 6, REV.
FIG. 4: detecting the contaminated FadV in the vaccine by using the RAA-LFD method; in the figure, 1-6 are respectively 104 TCID 501000 feather, 103 TCID 501000 feather, 102 TCID 501000 feather, 10TCID 501000 feather, 1TCID 501000 feather, 0.1TCID 501000 feather parts; 7 is PBS blank control; and 8 is a negative control.
FIG. 5: detecting the contaminated FadV in the vaccine by using a common PCR method; in the figure, 1-6 are respectively 104 TCID 501000 feather, 103 TCID 501000 feather, 102 TCID 501000 feather, 10TCID 501000 feather, 1TCID 501000 feather, 0.1TCID 501000 feather parts; 7 is PBS blank control; and 8 is a negative control.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.
As described in the background, the FAdV group I is most pathogenic and causes the greatest economic loss. Group I FAdV was subdivided into 5 subgroups (FAdV A-FAdV E), 12 serotypes (FAdV-1 to 7, FAdV-8a, FAdV-8b, FAdV-9 to 11). Prior art detection of FadV has focused primarily on serum type 4 FAdV (FAdV-4), serum type 8a FAdV (FAdV-8a), and serum type 8b FAdV (FAdV-8 b). However, for the purification of group I FadV, if 12 serotypes of group I FadV cannot be simultaneously detected, detection is likely to be missed.
Patent CN106868212A discloses a general PCR detection primer for subgroup I avian adenovirus, which is alleged that 494bp products can be amplified from 12 serotype viruses of subgroup I avian adenovirus theoretically. However, it only validated the results of the detection of 6 serotypes; the adopted method is a conventional PCR method, and gel electrophoresis analysis is required for result verification, so that the method is long in time consumption, complex in operation and high in requirements on instruments and equipment, and is not suitable for large-scale detection of chicken flocks in basic-level farms.
Based on the requirements of the RAA-LFD method on primer and probe design, the invention designs a pair of primers and probes in a conserved region of 12 different serotype FAdV reference sequences. Wherein the designed upstream primer sequence is as follows: 5 '-AGGTCCTGTTCGAAGAGGATDCGCGTGGTR-3', the sequence of the downstream primer is as follows: 5 '- [ Biotin ] GAHTACGTCAGCAAAAACATCGCVGCCAA-3', the pair of primers can amplify 152bp fragments of different serotype FAdV genomes; the designed probe sequence is TGTTGGTGGCGAAGGCGCCGTAGAGGGCGT [ FAM-dT ] "THF" C [ BHQ1-dT ] GAGCATTTGGAGATGG-C3 spacer. The single-tube reaction system is as follows: 1 tube of reaction dry powder, A Buffer (40.9. mu.L), upstream primer (2.0. mu.L), downstream primer (2.0. mu.L), probe (0.6. mu.L), sample (2.0. mu.L), B Buffer (2.5. mu.L), and the total volume of the reaction is 50. mu.L. After the amplification system is configured, fully oscillating and uniformly mixing, reacting for 8-15min in water bath at 39 ℃ after centrifugation, dropwise adding 10 mu L of RAA amplification product on a disposable nucleic acid test strip, inserting the test strip into an EP (ultraviolet) tube containing 100 mu L of buffer solution, and observing the result after 3 min. When 2 red bands appear (one is in the quality control area, and the other is in the test area), the result is positive, and the sample contains the nucleic acid fragment to be detected; if only one red band (in the quality control region) appears, the result is negative, which indicates that the fragment to be detected is not contained therein.
The invention can detect FadV of 12 different serotypes simultaneously; in addition, the method only needs necessary reagents and isothermal amplification equipment (such as a water bath kettle), is simple and convenient to operate, and is particularly suitable for large-scale detection of the chicken flocks in the basic farm, so that the purification of the FadV of the chicken flocks is realized.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention, which were not specifically described, were all those conventional in the art and commercially available. In the examples of the present invention, the specific experimental conditions and methods are not specified, and the conventional conditions such as J. SummBruker et al, science publishers, 2002, molecular cloning guidelines (third edition); master catalog of speekt et al, scientific press, 2001, cell experimental guidelines; or according to conditions recommended by the manufacturer.
The RAA nucleic acid amplification kit used in the present invention was purchased from Hangzhou scientific and biological sciences Co.
Example 1: optimization and establishment of RAA-LFD detection method
Design of RAA primers and probes
The entire amplification process of the RAA method is carried out under the conditions of normal temperature and constant temperature. The design of primers and probes is the key of the method, and the effectiveness, specificity and sensitivity of the method are directly influenced.
In terms of primer design, the general requirements of the RAA method are: (1) length of the primer: 30-36 bp; (2) the GC content is between 40 and 60 percent; (3) primer dimer and hairpin structures are avoided; (4) the size of the amplification product is 100-200 bp optimally, and is recommended not to exceed 500 bp.
However, the design goal of the present invention is to simultaneously detect 12 different serotypes of FadV, and the area that can be targeted is very limited, which is difficult for RAA primer design. In order to solve the difficulty, the invention also takes the following aspects into consideration on the basis of meeting the general requirements of RAA primer design:
(1) and (3) selecting a target area:
the invention selects the DNA polymerase gene of FadV as a target region to carry out RAA primer design. The gene codes virus DNA polymerase, which takes virus parental DNA as a template to catalyze substrate dNTP molecule polymerization to form filial DNA, plays an important role in FadV replication and is quite conservative in 12 different serotypes of FadV. Combining the RAA primer design requirement, finally determining 1277-1428bp of the DNA polymerase gene as the RAA primer amplification region, and the amplification length is 152 bp.
(2) Design of "degenerate primers":
degenerate primers are intended to mean mixtures of primers representing the possibility of encoding all different base sequences of a single amino acid. The lower the degeneracy, the stronger the product specificity; the higher the degree of degeneracy, the better the product sensitivity. Although DNA polymerases have high homology between FadV of different serotypes, there is little difference at each site. In order to enhance the sensitivity of the method, the scheme introduces a 'degenerate primer' in the primer, and other letters such as D, R, H, V (wherein D is A/G/T, R is A/G, H is A/C/T, and V is A/C/G) are also presented in the nucleotide chain besides the normal A, T, G, C four-base symbol. It should be noted that the primers should be designed so as to select amino acids with small degeneracy as much as possible and avoid degeneracy of the 3' -end of the primer.
The sequence of the finally designed upstream primer is as follows: 5 '-AGGTCCTGTTCGAAGAGGATDCGCGTGGTR-3', the sequence of the downstream primer is as follows: 5 '- [ Biotin ] GAHTACGTCAGCAAAAACATCGCVGCCAA-3'.
In terms of probe design, the probe design requirements mainly include: (1) a one base nucleotide like site, such as Tetrahydrofuran (THF); (2) a fluorescent group and a quenching group which are respectively positioned at two sides of THF; (3) 3' end modification (commonly used C3-spacer) to block probe extension; (4) the modifying group must be located on a thymine and the ideal probe position is generally limited to sequences with fewer than 5 nucleotides between two thymines.
From the above requirements, it can be seen that the probe not only has requirements on the sequence length and G/C content, but also has requirements on certain base types, modification types and positions of the base types in the sequence itself, so that the initial screening in the probe screening process can only be carried out by reading the base. As the experimental instrument used for exploring the optimal primer, probe concentration and optimal reaction temperature is a fluorescent gene detector produced by Hangzhou Zhongzhi testing Biotechnology Limited, the fluorescent modifying group is selected as FAM and the fluorescent quenching group is selected as BHQ 1. The method for modifying the probe comprises the following steps: the fluorescent reporter group is modified on the position of 31bp away from the 5' end base number of the probe sequence; the fluorescence quenching group is modified on the position of the probe sequence, which is 17bp away from the 3' end base number, 1 base C is arranged between the fluorescence reporting group and the quenching group, and the base C is replaced by a tetrahydrofuran residue; the probe sequence was finally determined to be: TGTTGGTGGCGAAGGCGCCGTAGAGGGCGT [ FAM-dT ] "THF" C [ BHQ1-dT ] GAGCATTTGGAGATGG-C3 spacer.
Optimization of RAA-LFD detection System
After the RAA primer and the probe are obtained through optimization design, the RAA-LFD detection system is further optimized, and the method specifically comprises the following steps:
(1) and (3) RAA-LFD primer probe concentration screening:
preparing a reaction solution: 40.9 mu L of A buffer, 2.5 mu L of B buffer, 2 mu L of template, 2 mu L of upstream primer, 2 mu L of downstream primer and 0.6 mu L of probe (the concentrations of the primer and the probe are respectively 1 mu M, 5 mu M, 10 mu M, 20 mu M and 50 mu M) are respectively added into a centrifuge tube and fully mixed to obtain a uniformly mixed reaction solution. And (3) putting the uniformly mixed reaction tube into a constant-temperature fluorescent gene detector, setting the reaction temperature to be 39 ℃, and reacting for 10 min. And (4) judging whether the amplification curve is positive or negative according to a positive judgment method in a detection instrument and the existence of an obvious amplification curve. The detection result shows that when the concentration of the primer probe is 0.5 mu M, the fluorescence value of the amplification curve is higher, and the peak emergence time is earlier, so that the concentration of the primer probe is 0.5 mu M in the experiment.
| |
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| Upstream primer | 0.1μM | 0.25μM | 0.5μM | 1μM | 2μM |
| Downstream primer | 0.1μM | 0.25μM | 0.5μM | 1μM | 2μM |
| Probe needle | 0.1μM | 0.25μM | 0.5μM | 1μM | 2μM |
(2) Optimizing the amplification time of the RAA-LFD identification method:
the reaction solution was prepared as described above. Placing the mixed reaction tube into a constant temperature fluorescent gene detector, and setting the reaction temperature at 38 deg.C, 39 deg.C, 40 deg.C, 41 deg.C, and the reaction time for 10 min. And (4) judging whether the amplification curve is positive or negative according to a positive judgment method in a detection instrument and the existence of an obvious amplification curve. The detection result shows that when the reaction temperature is 39 ℃, the fluorescence value of the amplification curve is higher, and the peak time is earlier, so that 39 ℃ is selected as the reaction temperature in the experiment.
After optimizing the reaction conditions and the system of the RAA-LFD detection method, the optimal reaction system conditions of the RAA-LFD detection method are finally determined as follows: the concentrations of the primers and the probes were 0.5. mu.M, and the reaction was carried out at 39 ℃ for 10 min.
Example 2: methodology investigation of RAA-LFD detection method
1. Sensitivity investigation:
carrying out PCR amplification by taking DNA of 12 different serotypes of FAdV as a template, wherein the upstream primer is as follows: 5 '-AGGTCCTGTTCGAAGAGGATDCGCGTGGTR-3', the downstream primer is 5 '-GAHTACGTCAGCAAAAACATCGCVGCCAA-3', the 12 fragments are connected with the pMD-18T vector, and 12 plasmid standard products carrying the target site gene sequences are constructed. The constructed 12 standards (copy number is 10)10Multiple copies) are diluted to 1 copy according to 10 times of gradient in turn, 10 copies are selected7One copy-1 copy of the product was used as a sample for RAA-LFD detection, and blank double distilled water was used as a negative control. Meanwhile, the same primers are used for carrying out common PCR amplification on 12 plasmid standard substances, and the sensitivity of the RAA-LFD method and the sensitivity of the common PCR method are compared. The results showed that the sensitivity of the established RAA-LFD method was 100 copies (FIG. 1), whereas the sensitivity of the conventional PCR was 1000 copies at the lowest, which was 10 times that of the conventional PCR (FIG. 2).
2. And (3) specific investigation:
respectively using DNA or cDNA of ALV-A, ALV-J, CIAV, MDV and REV stored in the laboratory as template and FAdV-C4 type 102Amplification comparison is carried out on the copies/mu L plasmid template, and the RAA-LFD detection system optimized in the embodiment 1 of the invention is adopted for detection.
The result is shown in fig. 3, removing the FAdV plasmid has a distinct red band in the detection zone, and the remaining sample detection zones have no red band and have a band in the quality control line, indicating that the test is established and has no cross reaction with other viruses, indicating that the RAA-LFD detection method of the present invention has good specificity.
Example 3: and (3) detecting a clinical sample:
the content of serum type 4 FAdV isolate FAdV-N22 was quantified by Reed-Muench method, and FAdV-N22 to 10 was diluted with PBS, respectively4TCID50/1ml、103TCID50/1ml、102TCID50/1ml、10TCID50/1ml、1TCID50/1ml、0.1TCID501ml of the mixed virus is subjected to 6 gradients, and then the mixed virus PBS is diluted into 6 bottles of live vaccine (1000 feathers) for the same batch of the newcastle disease, so that 10 bottles of separately artificially contaminated newcastle disease are obtained4 TCID 501000 feather, 103 TCID 501000 feather, 102 TCID 501000 feather, 10TCID 501000 feather, 1TCID 501000 feather, 0.1TCID50Per 1000 feathers of FAdV 6 bottles of live Newcastle disease vaccine (1000 feathers), and 1 bottle of vaccine was diluted with 1ml of sterile PBS as a blank control.
DNA is extracted from the 7 bottles of different vaccines by a virus nucleic acid extraction kit, and the RAA-LFD method and the common PCR method optimized in the embodiment 1 of the invention are respectively used for detecting the virus in the artificial simulated vaccine pollution. The results are shown in FIGS. 4 and 5.
The results show that: 1TCID can be detected by adopting RAA-LFD detection method50Vaccine contamination of 1000 feathers, while the common PCR method can detect vaccine contamination of 100 TCID50/1000 feathers at the lowest,
the above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> Shandong university of agriculture
<120> RAA primer for detecting 12 serotypes of avian adenovirus group I and detection method thereof
<130> 2021
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 30
<212> DNA
<213> Artificial sequence
<400> 1
aggtcctgtt cgaagaggat dcgcgtggtr 30
<210> 2
<211> 29
<212> DNA
<213> Artificial sequence
<400> 2
gahtacgtca gcaaaaacat cgcvgccaa 29
<210> 3
<211> 47
<212> DNA
<213> Artificial sequence
<400> 3
tgttggtggc gaaggcgccg tagagggcgt cgagcatttg gagatgg 47
Claims (8)
1. A RAA primer and probe combination for detecting 12 serotypes of avian adenovirus group I is characterized in that sequences of a primer and a probe in the RAA primer and probe combination are respectively shown as SEQ ID NO.1-SEQ ID NO. 3.
2. Use of the RAA primer and probe combination of claim 1 in the preparation of a reagent or kit for the detection of 12 serotypes of avian adenovirus group I.
3. A kit for detecting 12 serotypes of avian adenovirus group I, comprising the RAA primer and probe combination of claim 1.
4. The kit according to claim 3, further comprising: RAA reagent and lateral flow chromatography test strip.
5. The kit of claim 4, wherein the RAA-reactive reagent comprises: a buffer and B buffer.
6. A method for detecting 12 serotypes of avian adenovirus group I using a kit according to any one of claims 3 to 5, comprising the steps of:
and extracting DNA of a sample to be detected as a template, carrying out RAA amplification by using an RAA primer and probe combination in the kit, and detecting an RAA amplification product by using a lateral flow chromatography test strip.
7. The method of claim 6, wherein the reaction system for RAA amplification is: a buffer 40.9 μ L; b, 2.5 mu L of buffer; 2 mu L of upstream primer; 2 mu L of downstream primer; probe 0.6 μ L; template 2. mu.L.
8. The method of claim 6, wherein the RAA amplification conditions are: reacting for 8-15min at the constant temperature of 39 ℃.
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| CN114959120A (en) * | 2022-06-30 | 2022-08-30 | 华南农业大学 | Primer probe set and kit for detecting chicken infectious anemia virus by RAA fluorescence method and application of primer probe set and kit |
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Application publication date: 20211029 |