CN113981145B - Detection method and detection kit for group I avian adenovirus A, B, C, D and E - Google Patents
Detection method and detection kit for group I avian adenovirus A, B, C, D and E Download PDFInfo
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Abstract
The invention discloses a detection method and a detection kit for group I avian adenovirus A, B, C, D and E. The specific primer group and the probe group are designed, so that the poultry adenovirus group I A, B, C, D and the poultry adenovirus group I E can be rapidly and accurately detected and identified in one reaction. The detection kit obtained based on the design has stronger specificity and higher sensitivity, and can effectively detect the concentration of 2.1 multiplied by 10 2 The copies/mL virus does not need repeated detection, has high actual application accuracy, and can be widely used for monitoring, preventing and controlling the avian adenovirus infection epidemic situation.
Description
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a detection method and a detection kit for group I avian adenovirus A, B, C, D and E.
Background
The avian adenovirus I has A, B, C, D and E five species, wherein the A species comprises FADV-1, the B species comprises FADV-5, the C species comprises FADV-4 and FADV-10, the D species comprises FADV-2, FADV-3, FADV-9 and FADV-11, and the E species comprises FADV-6, FADV-7, FADV-8a and FADV-8B. A, C, D and E species are mainly popular in China, and FADV-4 has high toxicity and can cause Ankara disease or chicken pericardial effusion syndrome. In addition, infection with certain serotypes can also cause mild respiratory symptoms, egg drop, inclusion body hepatitis, myogastric erosion, immunosuppression and the like in poultry populations, so that growth of the poultry populations is hindered and mortality of the poultry populations is increased. Therefore, group I avian adenoviruses cause huge losses to the breeding plants.
Currently, detection of the avian adenovirus group I is mainly general detection or detection mainly focused on FADV-4, FADV-8a and FADV-8b, detection differentiation of the avian adenovirus group I cannot be carried out, and a method for simultaneously carrying out multiplex fluorescence PCR detection and identification on A, B, C, D and E five kinds of avian adenovirus group I is not disclosed.
Disclosure of Invention
The present invention is directed to solving at least one of the problems of the prior art described above. Therefore, the invention provides a detection method and a detection kit for the group I avian adenovirus A, B, C, D and the E avian adenovirus, which can detect and identify the group I avian adenovirus A, B, C, D and the E avian adenovirus by one-time PCR amplification and have stronger specificity and sensitivity.
In a first aspect of the invention, a multiplex fluorescent quantitative PCR primer set for detecting and identifying avian group I adenovirus is provided, wherein the avian group I adenovirus comprises A, B, C, D avian group I adenovirus and E avian group I adenovirus.
According to the first aspect of the invention, in some embodiments of the invention, the nucleotide sequences of the primer set are:
primers for avian group I adenovirus A, B, C, D and species E were identical and the sequences were as follows:
FADV-F：5’-CGAGATGGCGTACATGATCG-3’(SEQ ID NO.1)；
FADV-R：5’-CCTGGGTCAAACCGAACATG-3’(SEQ ID NO.2)。
in a second aspect of the invention, a multiplex fluorescent quantitative PCR probe set for simultaneously detecting and identifying avian group I adenovirus, wherein the avian group I adenovirus comprises A, B, C, D avian group I adenovirus and E avian group I adenovirus.
According to the second aspect of the invention, in some embodiments of the invention, the nucleotide sequence of the probe set is:
group I avian adenovirus A:
FADV-A-P：5’-CAGCAGCTGTCGGCCATCTGCA-3’(SEQ ID NO.3)；
group I avian adenovirus B:
FADV-B-P：5’-CCTGCACGCCCAAATCCAGCA-3’(SEQ ID NO.4)；
group I avian adenovirus C:
FADV-C-P：5’-CTACCGGGTGATGCAGGCTGTGCT-3’(SEQ ID NO.5)；
group I avian adenovirus D:
FADV-D-P：5’-ACCGTCCGGTGCGGTTCCGT-3’(SEQ ID NO.6)；
group I avian adenovirus E:
FADV-E-P：5’-CACCTGCACGCCCAAGTCTAACACG-3’(SEQ ID NO.7)；
or a nucleotide complement of such sequences.
In some preferred embodiments of the present invention, the two ends of the sequence of the probe set are labeled with a fluorescent group and a quencher group respectively.
In some more preferred embodiments of the present invention, the fluorescent group and the quencher group labeled with each probe sequence are different from each other.
In some more preferred embodiments of the invention, the FADV-A and FADV-B probe sequences are labeled with FAM as fluorescent groups and BHQ1 as quenching groups; the fluorescent group marked by the FADV-C probe sequence is TXR, the quenching group is BHQ2, the fluorescent group marked by the FADV-D probe sequence is CY5, the quenching group is BHQ2, the fluorescent group marked by the FADV-E probe sequence is VIC, and the quenching group is BHQ 2.
In a third aspect of the invention, a multiple fluorescent quantitative PCR reagent of avian adenovirus group I is provided, wherein the avian adenovirus group I comprises A, B, C, D avian adenovirus group I and E avian adenovirus group I.
According to the third aspect of the present invention, in some embodiments of the present invention, the PCR reagents comprise the primer set of the first aspect of the present invention and/or the probe set of the second aspect of the present invention.
In a fourth aspect of the invention, the invention provides an application of the PCR reagent in the third aspect of the invention in preparing a kit for detecting and identifying the group I avian adenovirus, wherein the group I adenovirus comprises A, B, C, D avian adenovirus group I and E avian adenovirus group I.
According to a fourth aspect of the present invention, in some embodiments of the present invention, the group I avian adenovirus detection and identification kit further comprises at least one of a PCR reaction solution, an enzyme, a negative quality control substance, and a positive quality control substance.
In some preferred embodiments of the present invention, the PCR reaction solution comprises 100-300 mM Tris, 100-300 mM KCl, 10-30 mM MgCl 2 0.03-0.05% of Tween-80 (Tween-80), 4.5-5.0% of glycerol, 0.1-5 mM dNTP and 10-20 mM (NH) 4 ) 2 SO 4 And 0.1% to 0.3% dimethyl sulfoxide (DMSO).
In some more preferred embodiments of the invention, the PCR reaction contains 150mM Tris, 100mM KCl, 15mM MgCl 2 0.03% Tween-80, 4.5% glycerol, 2.5mM dNTP, 10mM (NH) 4 ) 2 SO 4 And 0.15% DMSO.
In some preferred embodiments of the invention, the enzyme solution consists of 2U/. mu.L of hot start Taq enzyme, 25mM Tris, 15% glycerol and 10mM Dithiothreitol (DTT).
In some preferred embodiments of the invention, the negative quality control material is double distilled water.
In some preferred embodiments of the present invention, the positive quality control product is a standard strain A, B, C, D and E species amplified by primers of group I avian adenovirus A, B, C, D and E species, the target fragment is obtained by PCR amplification of the above primer set, and then 5 target fragments are constructed into a plasmid vector by a multi-fragment recombination method, thereby obtaining a positive expression plasmid.
In some preferred embodiments of the invention, the use of the kit comprises the steps of:
(1) extracting nucleic acid in a detection sample;
(2) performing PCR amplification by using a primer group and a probe group in the kit for detecting and identifying the group I avian adenovirus;
(3) and (3) qualitatively or quantitatively determining the group I avian adenovirus in the sample according to the amplification curve.
The quantitative detection method comprises the following steps: and (3) using the positive standard substance as positive quality control, drawing a standard curve, and calculating the concentration content of the positive quality control substance in the sample according to the CT value in the detection sample amplification curve.
In some more preferred embodiments of the present invention, the multiplex quantitative PCR amplification reaction system of the present invention is:
and
in some more preferred embodiments of the present invention, the multiplex quantitative PCR amplification reaction procedure of the present invention is: 93-95 ℃ for 5-15 min; at the temperature of 93-95 ℃ for 15-45 s; and (4) performing 40 cycles at 45-75 ℃ for 15-45 s.
The invention has the beneficial effects that:
(1) the invention breaks through the limitation of a common PCR method, A, B, C, D and E types of the avian adenovirus I can be detected by one-time PCR amplification, the specificity is strong, the types of the avian adenovirus I in a sample can be distinguished according to a fluorescence signal value, and an upstream primer and a downstream primer in an amplification system of the avian adenovirus I A, B, C, D and E are universal, so that the use of the primers is reduced, and the complementary interference of a plurality of pairs of primers in one system is avoided, and the amplification efficiency is influenced.
(2) The different primer probes have no complementary structures, and the annealing temperatures are close, so that the amplification efficiency of A, B, C, D of the avian adenovirus group I and the amplification efficiency of E of the avian adenovirus group I in the whole system are consistent.
(3) The detection method has high sensitivity, and the detection limit is 2.1x10 2 The copies/mL can be widely used for classifying and identifying the avian adenovirus in various detection samples, and provides favorable support for subsequent treatment work.
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FIG. 1 shows the detection results of the specific detection of FADV-A, FADV-C, FADV-D and FADV-E using the multiplex fluorescent quantitative PCR detection method in the examples of the present invention;
FIG. 2 is a diagram showing the detection results of the specific detection of FADV-B, FADV-C, FADV-D and FADV-E using the multiplex fluorescent quantitative PCR detection method in the present embodiment;
FIG. 3 shows the results of multiple fluorescent quantitative PCR assays for H9 subtype avian influenza, Newcastle disease virus, infectious bursal disease virus, duck plague virus, reticuloendotheliosis virus, avian reovirus, Marek's virus and negative controls using the multiplex fluorescent quantitative PCR assay in an example of the present invention;
FIG. 4 is a result of a sensitivity test of the multiplex quantitative PCR detection kit for FADV-A according to an embodiment of the present invention, wherein the concentration of FADV-A is 2.1 × 10 from left to right 6 、2.1×10 5 、2.1×10 4 、2.1×10 3 、2.1×10 2 And 2.1X10 1 copies/mL；
FIG. 5 is a sensitivity test result of the multiple fluorescent quantitative PCR detection kit to FADV-B in the embodiment of the invention, wherein the concentration of FADV-B is 2.1 × 10 from left to right in sequence 6 、2.1×10 5 、2.1×10 4 、2.1×10 3 、2.1×10 2 And 2.1X10 1 copies/mL；
FIG. 6 is a result of a sensitivity test of the multiplex quantitative PCR detection kit for FADV-C according to an embodiment of the present invention, wherein the concentration of FADV-C is 2.1 × 10 from left to right 6 、2.1×10 5 、2.1×10 4 、2.1×10 3 、2.1×10 2 And 2.1X10 1 copies/mL；
FIG. 7 is a result of a sensitivity test of the multiplex quantitative PCR detection kit for FADV-D according to an embodiment of the present invention, wherein the concentration of FADV-D is 2.1 × 10 from left to right 6 、2.1×10 5 、2.1×10 4 、2.1×10 3 、2.1×10 2 And 2.1X10 1 copies/mL；
FIG. 8 is a result of a sensitivity test of the multiplex quantitative PCR detection kit for FADV-E according to an embodiment of the present invention, wherein the concentration of FADV-E is 2.1 × 10 from left to right 6 、2.1×10 5 、2.1×10 4 、2.1×10 3 、2.1×10 2 And 2.1X10 1 copies/mL；
FIG. 9 is a result of detecting 1 FADV-A infected avian thymus tissue by the multiplex quantitative PCR detection kit in the embodiment of the present invention, wherein the line A is an amplification curve of the FADV-A infected avian thymus tissue, and the line B is a negative quality control line;
FIG. 10 is a detection result of the multiplex quantitative PCR detection kit of the present invention on 1 FADV-5 (belonging to FADV-B) cell culture fluid, wherein the A line is an amplification curve of the FADV-5 (belonging to FADV-B) cell culture fluid, and the B line is a negative quality control line;
FIG. 11 is a detection result of the multiplex quantitative PCR detection kit of the present invention on 1 FADV-4 (FADV-C) cell culture fluid, wherein the line A is an amplification curve of the FADV-4 (FADV-C) cell culture fluid, and the line B is a negative quality control line;
FIG. 12 is a graph showing the detection result of the multiplex fluorescence quantitative PCR detection kit in the embodiment of the present invention on 1 FADV-9 (belonging to FADV-D) infected avian small intestine, wherein the line A is the amplification curve of FADV-9 (belonging to FADV-D) infected avian small intestine, and the line B is the negative quality control line;
FIG. 13 is a detection result of a multiple fluorescent quantitative PCR detection kit in an embodiment of the present invention on a kidney tissue sample infected with FADV-6 (belonging to FADV-E), FADV-7 (belonging to FADV-E) and FADV-8B (belonging to FADV-E), where an A line is a detection result of a kidney tissue sample infected with FADV-7 (belonging to FADV-E), a B line is a detection result of a kidney tissue sample infected with FADV-6 (belonging to FADV-E), a C line is a detection result of a kidney tissue sample infected with FADV-8B (belonging to FADV-E), and a D line is a negative quality control line.
Detailed Description
The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.
Preparation of multiple fluorescent quantitative PCR kit for detecting and identifying group I avian adenovirus
(1) Designing and synthesizing a primer probe:
according to the genomic sequences of A, B, C, D and E5 viruses of avian adenovirus group I published by Genebank, 5 groups of virus genome sequences are aligned and analyzed, probes are designed according to specific regions of each virus, and an upstream primer and a downstream primer are designed according to conserved regions of each virus. And (3) testing and screening the designed primers and probes one by one, and selecting the optimal primer-probe combination.
Wherein, the fluorescent groups marked by the FADV-A and FADV-B probe sequences are FAM; the fluorescent group marked by the FADV-C probe sequence is TXR, the fluorescent group marked by the FADV-D probe sequence is CY5, and the fluorescent group marked by the FADV-E probe sequence is VIC.
The primer pairs obtained by final screening and the corresponding probe sequences are as follows:
the sequences of the upstream primer and the downstream primer and the probe of the avian adenovirus group I A are respectively as follows:
an upstream primer: FADV-F: 5'-CGAGATGGCGTACATGATCG-3' (SEQ ID NO. 1);
a downstream primer: FADV-R: 5'-CCTGGGTCAAACCGAACATG-3' (SEQ ID NO. 2);
and (3) probe: FADV-A-P: 5'-CAGCAGCTGTCGGCCATCTGCA-3' (SEQ ID NO. 3);
the sequences of the upstream primer and the downstream primer and the probe of the group I avian adenovirus B are respectively as follows:
an upstream primer: FADV-F: 5'-CGAGATGGCGTACATGATCG-3' (SEQ ID NO. 1);
a downstream primer: FADV-R: 5'-CCTGGGTCAAACCGAACATG-3' (SEQ ID NO. 2);
and (3) probe: FADV-B-P: 5'-CCTGCACGCCCAAATCCAGCA-3' (SEQ ID NO. 4);
the sequences of the upstream primer and the downstream primer and the probe of the avian adenovirus group I C are respectively as follows:
an upstream primer: FADV-F: 5'-CGAGATGGCGTACATGATCG-3' (SEQ ID NO. 1);
a downstream primer: FADV-R: 5'-CCTGGGTCAAACCGAACATG-3' (SEQ ID NO. 2);
and (3) probe: FADV-C-P: 5'-CTACCGGGTGATGCAGGCTGTGCT-3' (SEQ ID NO. 5);
the sequences of the upstream primer and the downstream primer and the probe of the group I avian adenovirus D are respectively as follows:
an upstream primer: FADV-F: 5'-CGAGATGGCGTACATGATCG-3' (SEQ ID NO. 1);
a downstream primer: FADV-R: 5'-CCTGGGTCAAACCGAACATG-3' (SEQ ID NO. 2);
and (3) probe: FADV-D-P: 5'-ACCGTCCGGTGCGGTTCCGT-3' (SEQ ID NO. 6);
the sequences of the upstream primer and the downstream primer and the probe of the avian adenovirus group I E are respectively as follows:
an upstream primer: FADV-F: 5'-CGAGATGGCGTACATGATCG-3' (SEQ ID NO. 1);
a downstream primer: FADV-R: 5'-CCTGGGTCAAACCGAACATG-3' (SEQ ID NO. 2;
and (3) probe: FADV-E-P: 5'-CACCTGCACGCCCAAGTCTAACACG-3' (SEQ ID NO. 7).
(2) Preparing a multiple fluorescent quantitative PCR amplification system:
a multiplex quantitative PCR amplification system was prepared based on the systems shown in tables 1 and 2 (the amplification was carried out in two systems (System 1 and System 2) because of the restriction of PCR amplification capacity, and naturally, the amplification could be carried out in the same system under the condition that the capacity was allowed).
TABLE 1 multiplex fluorescent quantitative PCR amplification System 1
| Components | Content (wt.) |
| PCR reaction solution | 18.71μL |
| FADV-F | 0.4μL |
| FADV-R | 0.4μL |
| FADV-A-P | 0.1μL |
| FADV-C-P | 0.12μL |
| FADV-D-P | 0.12μL |
| FADV-E-P | 0.15μL |
| Enzyme | 1μL |
| Detection sampleProduct nucleic acid | 4μL |
TABLE 2 multiplex fluorescent quantitative PCR amplification System 2
| Components | Content (wt.) |
| PCR reaction solution | 18.71μL |
| FADV-F | 0.4μL |
| FADV-R | 0.4μL |
| FADV-B-P | 0.1μL |
| FADV-C-P | 0.12μL |
| FADV-D-P | 0.12μL |
| FADV-E-P | 0.15μL |
| Enzyme | 1μL |
| Detecting sample nucleic acid | 4μL |
In this embodiment, the inventor tests to obtain an optimal combination of primer pairs and probe concentrations, where the concentration of each primer pair is 1 μ M, and the probe concentrations are different according to the types of viruses, specifically: the concentration of the FADV-A probe is 0.25 mu M, the concentration of the FADV-B probe is 0.25 mu M, the concentration of the FADV-C probe is 0.3 mu M, the concentration of the FADV-D probe is 0.3 mu M, and the concentration of the FADV-E probe is 0.38 mu M.
The nucleic acid of the sample to be tested can be extracted using a conventional nucleic acid extraction kit (a DNA extraction kit is used according to the type of the sample, or reverse transcription is performed after RNA is extracted using an RNA extraction kit).
The PCR reaction solution is a multiplex fluorescence PCR reaction solution that is conventional in the art, and in this embodiment, the specific components thereof are: 150mM Tris, 100mM KCl, 15mM MgCl 2 0.03% Tween-80, 4.5% glycerol, 2.5mM dNTP, 10mM (NH) 4 ) 2 SO 4 And 0.15% DMSO.
The enzyme is a kind of enzyme suitable for multiplex fluorescent PCR, which is conventional in the art, and in this example, it is specifically: 2U/. mu.L of hot start Taq enzyme, 25mM Tris, 30% glycerol and 1mM DTT. Wherein the optimal use concentration is obtained by screening the hot start Taq enzyme in a gradient change of 0.5U/. mu.L within the concentration range of 0.5-5U/. mu.L.
The kit in this embodiment may further contain a positive quality control material and a negative quality control material.
In this example, the positive quality control substances are: the standard strain is amplified by FADV-A, FADV-B, FADV-C, FADV-D and FADV-E primers, then a target fragment is obtained by utilizing the PCR amplification of the primer group in the embodiment, and 5 target fragments are constructed into a plasmid vector by utilizing a multi-fragment recombination mode, so that a positive expression plasmid is obtained.
In this embodiment, the negative quality control material is double distilled water.
The PCR amplification procedure in this example was: 93-95, 5-15 min; 93-95 m, 15-45 s; 45-75 s, 15-45 s, 40 cycles. In this example, the amplification program was set up as: 94 ℃ for 5 min; 30s at 94 ℃; 60 ℃, 30s, 40 cycles.
(3) Qualitative and quantitative determination of the virus in the sample:
and performing qualitative and quantitative analysis on the viruses in the sample according to the amplification curve.
The result judgment standard is as follows:
positive: the Ct value of the detected sample is less than or equal to 35.0, and the curve has obvious exponential growth period;
and (3) suspicious: the Ct value of the detection sample is more than 35.0 and less than 38.0, the experiment is repeated, if the Ct value is still less than 38.0 and the curve has obvious exponential growth period, the result is positive, otherwise, the result is negative;
negative: no sample Ct value was detected or Ct value was greater than 38.
Quantitative standard: and (3) using the positive standard substance as positive quality control, drawing a standard curve, and calculating the concentration content of the positive quality control substance in the sample according to the Ct value in the amplification curve of the detection sample.
Evaluation of detection Effect of detection and discrimination kit in the above-described examples
(1) And (3) specificity test:
and performing specificity test on the FADV-A, FADV-B, FADV-C, FADV-D, FADV-E standard sample, H9 subtype avian influenza, Newcastle disease virus, infectious bursal disease virus, duck plague virus, avian reticuloendotheliosis virus, avian reovirus, Marek's virus and a negative control group.
Detection was carried out according to the detection method described in the above examples (both the amplification system and the amplification procedure were kept in agreement). The detection results are shown in FIGS. 1 to 3. As can be seen from the results, the specific detection results of the FADV-A, FADV-B, FADV-C, FADV-D, FADV-E standard sample are all positive to the corresponding detection item; h9 subtype avian influenza, newcastle disease virus, infectious bursal disease virus, duck plague virus, reticuloendotheliosis virus, avian reovirus, Marek's virus and a negative control group have no amplification curve, and detection results are negative, which indicates that the multiple fluorescence quantitative PCR detection kit in the embodiment of the invention has better specificity.
(2) And (3) sensitivity detection:
the concentration of the nucleic acid of the positive quality control material (the preparation method is the same as the above example) is 3.5X 10 measured by an ultraviolet spectrophotometer 6 copies/mL, diluted 10-fold with double distilled water (concentrations after dilution of 2.1X10, respectively) 5 、2.1×10 4 、2.1×10 3 、2.1×10 2 And 2.1X10 1 copies/mL) were detected using the detection method described in the above example (both the amplification system and the amplification procedure were consistent).
The detection results are shown in FIGS. 4 to 8. FIGS. 4 to 8 are respectively a graph of the sensitivity of the detection method in the above embodiment for detecting fluorescence FAM in FADV-A, FADV-B, the sensitivity for detecting fluorescence TXR in FADV-C, the sensitivity for detecting fluorescence CY5 in FADV-D, and the sensitivity for detecting fluorescence VIC in FADV-E, where the sample concentration from left to right in FIGS. 4 to 8 is sequentially 2.1 × 10 6 、2.1×10 5 、2.1×10 4 、2.1×10 3 、2.1×10 2 、2.1×10 1 copies/mL, of which 2.1X10 can be found 1 copies/mL no amplification curve. Thus, it can be shown that the detection sensitivity of the detection method in the above example for FADV-A, FADV-B, FADV-C, FADV-D and FADV-E samples is as follows: 2.1X10 2 copies/mL。
Practical application effect of the detection kit in the above embodiment
In order to verify whether the detection and identification kit in the above embodiment can be practically applied to daily detection, the inventors selected common samples in daily detection to perform detection tests on the detection and identification kit in the above embodiment.
In this embodiment, the detection samples are brain, thymus, trachea, pancreas, bursa of fabricius, glandular stomach, caecum tonsil, small intestine, spleen, lung, kidney and liver, specifically: FADV-C infects spleen tissue 2 cases, FADV-4 (belonging to FADV-C) cell culture fluid 1 case, FADV-A infects thymus tissue 1 case, FADV-5 (belonging to FADV-B) cell culture fluid 1 case, FADV-6 (belonging to FADV-E) and FADV-7 (belonging to FADV-E) and FADV-8B (belonging to FADV-E) infect kidney tissue 1 case respectively, FADV-10 (belonging to FADV-C) infects kidney tissue 2 cases, FADV-9 (belonging to FADV-D) and FADV-10 (belonging to FADV-C) infect bird small intestine 1 case respectively. And H9 subtype avian influenza, Newcastle disease virus, infectious bursal disease virus, duck plague virus, reticuloendotheliosis virus, avian reovirus and Marek's virus samples are used as negative controls.
The specific detection steps are as follows:
(1) sample pretreatment:
tissue sample: grinding 0.5g in grinder, adding 1.5mL physiological saline, grinding, homogenizing, transferring to 1.5mL EP tube, centrifuging at 8000rpm for 2min, and collecting supernatant 200 μ L in 1.5mL EP tube. Other liquid samples: 200 μ L were taken directly for DNA/RNA extraction.
(2) Sample detection:
the treated sample was subjected to nucleic acid extraction using a nucleic acid extraction kit (purchased from QIAGEN). The extracted nucleic acid sample to be tested was tested according to the test methods described in the above examples (the amplification system was kept consistent).
Setting negative control and positive control (refer to the negative control and the positive control in the above embodiment), placing each reaction tube into a reaction tank of a quantitative PCR instrument, setting the name and the type of each detected fluorescent group, setting the report groups as FAM, VIC, TXR and CY5, selecting none as a quenching group, and carrying out amplification at 95 ℃ for 5 min; at 95 ℃ for 20 s; 55 ℃ for 40s, 40 cycles.
It is known that 2 cases of FADV-C infected chicken spleen tissue, 1 case of FADV-4 (belonging to FADV-C) cell culture fluid, 1 case of FADV-A infected chicken thymus tissue, 1 case of FADV-5 (belonging to FADV-B) cell culture fluid, 1 case of FADV-6 (belonging to FADV-E), 1 case of FADV-7 (belonging to FADV-E) and FADV-8B (belonging to FADV-E) infected kidney tissue, 2 cases of FADV-10 (belonging to FADV-C) infected kidney tissue, and 1 case of FADV-9 (belonging to FADV-D) and FADV-10 (belonging to FADV-C) infected chicken small intestine are positive. The experimental results are shown in fig. 9 to 13, and show that the known FADV-a and FADV-B positive samples detect the S-type curve in the FAM channel, the known FADV-C positive samples detect the S-type curve in the TXR channel, the known FADV-D positive samples detect the S-type curve in the CY5 channel, and the known FADV-E positive samples detect the S-type curve in the VIC channel. Therefore, the accuracy of detecting the target by the primer group and the probe group in the embodiment of the invention reaches 100%, and the invention has good specificity and good application effect.
The examples are a refinement and an explanation of the contents of the technical solution or an optimized technical solution, i.e. to one or several specific embodiments. The examples are to be construed as detailed and as specific as possible, and the invention will be embodied in such a manner that the production area, name, type, operation procedure, composition, formulation, use, effect, parameter, condition, means and the like of the raw materials can be clarified as much as possible.
SEQUENCE LISTING
<110> Guangzhou Weijie biological medicine science and technology Co Ltd
<120> detection method and detection kit for group I avian adenovirus A, B, C, D and E
<130>
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence
<400> 1
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence
<400> 2
<210> 3
<211> 22
<212> DNA
<213> Artificial sequence
<400> 3
cagcagctgt cggccatctg ca 22
<210> 4
<211> 21
<212> DNA
<213> Artificial sequence
<400> 4
cctgcacgcc caaatccagc a 21
<210> 5
<211> 24
<212> DNA
<213> Artificial sequence
<400> 5
ctaccgggtg atgcaggctg tgct 24
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence
<400> 6
<210> 7
<211> 25
<212> DNA
<213> Artificial sequence
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cacctgcacg cccaagtcta acacg 25
Claims (6)
1. A multiple fluorescence quantitative PCR primer group and probe group for detecting and identifying poultry adenovirus group I is characterized in that the poultry adenovirus group I comprises poultry adenovirus group I A, B, C, D and E species;
the nucleotide sequence of the primer group is as follows:
primers for avian group I adenovirus A, B, C, D and species E were identical and the sequences were as follows:
FADV-F：5’-CGAGATGGCGTACATGATCG-3’；
FADV-R：5’-CCTGGGTCAAACCGAACATG-3’；
the nucleotide sequence of the probe group is as follows:
group I avian adenovirus a probe:
FADV-A-P：5’-CAGCAGCTGTCGGCCATCTGCA-3’；
group I avian adenovirus B probe:
FADV-B-P：5’-CCTGCACGCCCAAATCCAGCA-3’；
group I avian adenovirus C probe:
FADV-C-P：5’-CTACCGGGTGATGCAGGCTGTGCT-3’；
group I avian adenovirus D probe:
FADV-D-P：5’-ACCGTCCGGTGCGGTTCCGT-3’；
group I avian adenovirus E probe:
FADV-E-P：5’-CACCTGCACGCCCAAGTCTAACACG-3’。
2. the primer group and the probe group according to claim 1, wherein the two ends of the sequence of the probe group are respectively marked with a fluorescent group and a quenching group; the fluorescent group and the quenching group marked by each probe sequence are different.
3. A multiplex fluorescence quantitative PCR reagent for detecting and identifying avian adenovirus group I, which contains the primer group and the probe group of claim 1.
4. The use of the multiplex fluorescent quantitative PCR reagent of claim 3 in the preparation of a group I avian adenovirus detection and/or identification kit.
5. The use according to claim 4, wherein the kit for detecting and/or identifying group I avian adenovirus further comprises at least one of a PCR reaction solution, an enzyme, a negative quality control substance and a positive quality control substance.
6. The use of claim 4, wherein said avian group I adenovirus comprises avian group I adenovirus A, B, C, D and E.
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Denomination of invention: A Detection Method and Kit for Group I Avian Adenovirus A, B, C, D, and E Species Effective date of registration: 20230609 Granted publication date: 20220812 Pledgee: Bank of China Limited by Share Ltd. Guangzhou Liwan branch Pledgor: Guangzhou Weijie Biomedical Technology Co.,Ltd. Registration number: Y2023980043580 |
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