CN107475462B - Kit for identifying common serotype chicken inclusion body hepatitis infection and application thereof - Google Patents
Kit for identifying common serotype chicken inclusion body hepatitis infection and application thereof Download PDFInfo
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention belongs to the technical field of biological detection, and particularly relates to a kit for identifying common serotype chicken inclusion body hepatitis infection and application thereof. The detection kit for identifying the common serotype chicken inclusion body hepatitis infection utilizes the characteristics of rapidness, accuracy and high sensitivity of a PCR technology, utilizes three pairs of designed specific primers to distinguish three domestic mainly popular chicken inclusion body hepatitis types of 8a, 8b and 11 in the same system, can quickly distinguish infection of chicken inclusion body hepatitis serum types of 8a, 8b and 11 or mixed infection of the viruses, is reported for the first time in clinic, has the advantages of time saving, labor saving, low cost and the like, and has important application value.
Description
Technical Field
The invention belongs to the technical field of biological detection, and particularly relates to a kit for identifying common serotype chicken inclusion body hepatitis infection and application thereof.
Background
Inclusion Body Hepatitis (IBH) is an acute infectious disease of chickens caused by avian adenovirus group I, mostly occurs in chickens of 2-5 weeks old, wherein the chicken is most frequently aged for 3-4 weeks, the main symptom is that the mortality of the chickens is suddenly increased, the morbidity reaches 10-20%, but the mortality is low and generally is not more than 5%, and the chicken usually returns to normal after 5-7 days of the morbidity. Infected sick chicken mainly shows the symptoms of mental depression, inappetence, lethargy, white cockscomb, disordered feather and the like; the autopsy shows that the inclusion body hepatitis which takes the yellowing, swelling and embrittlement of the liver as main pathological features rarely has pericardial effusion, part of the kidney is swollen and is light yellow, the spleen is also swollen, and the bone marrow is light red to light yellow; the most significant pathological change is inclusion bodies in the liver cell nucleus.
In recent years, the disease is popular and tends to rise in broiler chickens in China, and at present, two main diseases caused by avian adenovirus group I in China are: firstly, pericardial effusion-hepatitis syndrome, mainly caused by serum type 4; the other is inclusion body hepatitis, which is mainly caused by serum 8a (FAV-8a), 8b (FAV-8b) and 11 (FAV-11) viruses, but the 3 serotypes have very low cross protection and can not be distinguished from clinical symptoms and pathological changes, so that great difficulty is brought to the prevention and treatment of the disease, and a method for identifying common serotype chicken inclusion body hepatitis infection is urgently needed to improve the treatment pertinence of the inclusion body hepatitis.
Disclosure of Invention
Therefore, the technical problem to be solved by the invention is to provide a kit for identifying common serotype inclusion body hepatitis infection so as to solve the problem that inclusion body hepatitis is difficult to distinguish in production.
The second technical problem to be solved by the invention is to provide a method for identifying the infection of the inclusion body hepatitis of the common serotype chicken and application thereof
In order to solve the technical problems, the kit for identifying the common serotype chicken inclusion body hepatitis infection comprises the following components by 100 times of dosage:
PCR reaction system 4800. mu.L:
positive plasmid 200. mu.L;
the positive plasmid is a mixture of recombinant plasmids T-8a, T-8b and T-11 with the final concentration of 0.1 ng/. mu.L.
The PCR reaction solution system comprises the following components:
the detection primer FAV8a-1/FAV8a-2 is used for detecting the avian adenovirus group I of serum 8a type, and the amplified fragment size is 292 bp;
the detection primer FAV8a-1/FAV8a-2 has the following sequence structure:
FAV8a-1：5'-AACCCCTATGAGAATACCACT-3'；
FAV8a-2：5'-CCAAATATTTCGTGCTCCC-3'。
the detection primer FAV8b-1/FAV8b-2 is used for detecting the avian adenovirus group I of serum 8b type, and the size of an amplified fragment is 208 bp;
the detection primer FAV8b-1/FAV8b-2 has the following sequence structure:
FAV8b-1：5'-GCCAGCGTTTCAGGCTCT-3'；
FAV8b-2：5'-CGTAGTAAGGCGTTGTTCCA-3'。
the detection primer FAV11-1/FAV11-2 is used for detecting the avian adenovirus group I of the serotype 11, and the amplified fragment size is 474 bp;
the detection primers FAV11-1/FAV11-2 respectively have the following sequence structures:
FAV11-1：5'-GAAGACTTTAGCGCCTCG-3'；
FAV11-2：5'-CTCGGTTCCCTTATCACCC-3'。
the recombinant plasmids T-8a, T-8b and T-11 were prepared as follows: FAV-8a, FAV-8b and FAV11 virus nucleic acids are respectively extracted, primers FAV8a-1/FAV8a-2, FAV8b-1/FAV8b-2 and FAV11-1/FAV11-2 are respectively utilized to carry out positive PCR amplification on a target strip, the target strip is recovered and is connected with a vector pMD18-T, the target strip is transformed into DH5 alpha competent cells, plasmids are extracted, and sequencing is carried out after positive PCR identification; plasmids with correct sequences were labeled as T-8a, T-8b and T-11 and served as positive controls for FAV-8a, FAV-8b and FAV11 viruses, respectively.
The reaction system (50 μ L) for the positive PCR amplification step comprises the following components:
ddH2o38.5. mu.L, 10 XPCR buffer 5. mu.L, 2.5mM dNTP 3. mu.L, 5U/. mu.L Taq DNA polymerase 0.5. mu.L, 20 pmol/. mu.L upstream and downstream primers 0.5. mu.L, sample nucleic acid 2. mu.L;
the PCR amplification reaction program comprises the following steps: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 15sec, and elongation at 72 ℃ for 30 sec; extension at 72 ℃ for 5 min.
The invention also discloses a use method of the kit for identifying the common serotype chicken inclusion body hepatitis infection, which comprises the following steps:
(1) extracting nucleic acid of a sample: taking 200 mu L of pathological material homogenate to be detected, and extracting sample nucleic acid by using a virus nucleic acid extraction kit for later use;
(2) and (3) PCR reaction: and (3) taking 48 mu L of the PCR reaction solution and 2 mu L of the sample nucleic acid, mixing uniformly, and carrying out PCR reaction according to the following procedures: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 15sec, and elongation at 72 ℃ for 30 sec; extending for 5min at 72 ℃;
(3) positive control: and (3) taking 48 mu L of the PCR reaction solution, adding 2 mu L of the positive plasmid, uniformly mixing, and reacting according to the following procedure: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 15sec, and elongation at 72 ℃ for 30 sec; extending for 5min at 72 ℃;
(4) and (3) electrophoretic observation: respectively adding 5 mu L of the PCR product and the positive product into 1.5% agarose gel for electrophoresis observation, and if a 292bp band appears, judging that the FAV-8a virus is positive; if a band of 208bp appears, the FAV-8b virus is judged to be positive; if a 474bp strip appears, the FAV-11 virus is judged to be positive; if the above-mentioned bands appear mixedly, it is a mixed infection.
The invention also discloses application of the kit for identifying the common serotype chicken inclusion body hepatitis infection in identifying the common serotype chicken inclusion body hepatitis infection.
The invention also discloses a method for identifying common serotype chicken inclusion body hepatitis infection, which comprises the step of identifying by using the kit.
The detection kit for identifying the common serotype chicken inclusion body hepatitis infection utilizes the characteristics of rapidness, accuracy and high sensitivity of a PCR technology, utilizes three pairs of designed specific primers to distinguish three domestic mainly popular chicken inclusion body hepatitis types of 8a, 8b and 11 in the same system, can quickly distinguish infection of chicken inclusion body hepatitis serum types of 8a, 8b and 11 or mixed infection of the viruses, is reported for the first time in clinic, has the advantages of time saving, labor saving, low cost and the like, and has important application value.
The detection kit for identifying the common serotype chicken inclusion body hepatitis infection has the advantages of low minimum detection amount of less than 10pg for three serotype viruses, high sensitivity, good specificity and the like.
Drawings
In order that the present disclosure may be more readily and clearly understood, the following detailed description of the present disclosure is provided in connection with specific embodiments thereof and the accompanying drawings, in which,
FIG. 1 shows the specific detection results of the kit of the present invention; wherein M is DNA Marker DL2000, and the viruses corresponding to 1-10 are respectively 8a type I poultry adenovirus, 8b type I poultry adenovirus, 11 type I poultry adenovirus, 8a type I poultry adenovirus and 8b type I poultry adenovirus mixture, 8a type I poultry adenovirus and 11 type I poultry adenovirus mixture, 8b type I poultry adenovirus and 11 type I poultry adenovirus mixture, 8a type I poultry adenovirus, 8b type I poultry adenovirus and 11 type I poultry adenovirus mixture, 1 type I poultry adenovirus, 2 type I poultry adenovirus and 4 type I poultry adenovirus;
FIG. 2 is the result of the sensitivity test of the kit of the present invention to 8a type group I avian adenovirus; wherein M is DNA Marker DL2000, and the content of virus DNA corresponding to 1-7 is 17.2ng, 1.72ng, 172pg, 17.2pg, 1.72pg, 0.172pg and 0.0172pg respectively;
FIG. 3 shows the result of the sensitivity test of the kit of the present invention to 8b type group I avian adenovirus; wherein M is DNA Marker DL2000, and the content of virus DNA corresponding to 1-7 is respectively 8.6ng, 0.86ng, 86pg, 8.6pg, 0.86pg, 0.086pg and 0.0086 pg;
FIG. 4 shows the result of the sensitivity test of the kit of the present invention to type 11 avian adenovirus group I; wherein M is DNA Marker DL2000, the content of virus DNA corresponding to 1-7 is respectively 10.5ng, 1.05ng, 105pg, 10.5pg, 1.05pg, 0.105pg and 0.0105 pg;
FIG. 5 is a schematic diagram showing a repetitive test of the kit of the present invention for avian adenovirus group I type 8 a; wherein M is DNA Marker DL2000, and 1-3 corresponds to three repeated detection results;
FIG. 6 is a schematic diagram showing a repetitive test of the kit of the present invention for avian adenovirus group I8 b virus; wherein M is DNA Marker DL2000, and 1-3 corresponds to three repeated detection results;
FIG. 7 is a schematic representation of a reproducibility test of the kit of the invention for avian adenovirus group I type 11; wherein M is DNA Marker DL2000, and 1-3 corresponds to three repeated detection results.
Detailed Description
Example 1 design of detection primers
According to conserved sequences of 8a type, 8b type and 11 type I group poultry adenovirus Hexon genes registered in Genbank, a pair of primers are respectively designed by using Primer5.0 software and are used for detecting 8a type, 8b type and 11 type I group poultry adenovirus.
The primer for detecting the serum 8a type I group avian adenovirus is recorded as FAV8a-1/FAV8a-2, the size of an amplified fragment is 292bp, and the sequence structure comprises:
FAV8a-1 (upstream primer): 5'-AACCCCTATGAGAATACCACT-3', respectively;
FAV8a-2 (downstream primer): 5'-CCAAATATTTCGTGCTCCC-3' are provided.
The primer for detecting the serum 8b type I group avian adenovirus is marked as FAV8b-1/FAV8b-2, the size of an amplified fragment is 208bp, and the sequence structure comprises:
FAV8b-1 (upstream primer): 5'-GCCAGCGTTTCAGGCTCT-3', respectively;
FAV8b-2 (downstream primer): 5'-CGTAGTAAGGCGTTGTTCCA-3' are provided.
The primer for detecting the serum 11 type group I avian adenovirus is recorded as FAV11-1/FAV11-2, the size of an amplified fragment is 474bp, and the sequence structure comprises:
FAV11-1 (upstream primer): 5'-GAAGACTTTAGCGCCTCG-3', respectively;
FAV11-2 (downstream primer): 5'-CTCGGTTCCCTTATCACCC-3' are provided.
Example 2 specificity of multiplex PCR
Nucleic acids of avian adenovirus group I of serogroup 1, 2, 4, 8a, 8b and 11 were extracted according to the virus nucleic acid extraction kit instructions, and PCR reactions were performed using these as templates, respectively.
The PCR reaction system (50. mu.L) for each viral nucleic acid included: ddH2O38.5. mu.L, 10 XPCR buffer 5. mu.L, 2.5mM dNTP 3. mu.L, 5U/. mu.L Taq DNA polymerase 0.5. mu.L, 20 pmol/. mu.L upstream and downstream primers 0.5. mu.L, sample nucleic acid 2. mu.L. Placing the mixture in a PCR amplification instrument for reaction, wherein the reaction program comprises the following steps: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 15sec, and elongation at 72 ℃ for 30 sec; extension at 72 ℃ for 5 min.
After the reaction, 5. mu.L of the PCR product was applied to 1.5% agarose gel for electrophoresis and observed, the results are shown in FIG. 1. Therefore, the multiplex PCR method can amplify a 292bp band from 8a type virus, a 208bp band from 8b type virus, a 474bp band from 11 type virus, two 292bp and 208bp bands from 8a type and 8b type mixed virus, two 292bp and 474bp bands from 8a type and 11 type mixed virus, two 208bp and 474bp bands from 8b type and 11 type mixed virus, three 292bp, 208bp and 474bp bands from 8a type, 8b type and 11 type mixed virus and no band from 1 type, 2 type, 4 type and 5 type virus.
EXAMPLE 3 sensitivity of multiplex PCR
Nucleic acids of avian adenovirus group I, 8a, 8b and 11 in serum were extracted according to the instructions of a viral nucleic acid extraction kit (AXYGEN), the contents of DNAs were measured by a spectrophotometer, the DNAs were diluted 10-fold, and after PCR amplification reaction was completed under optimized reaction conditions, 5. mu.L of each of the PCR products was added to 1.5% agarose gel for electrophoresis and observed, and the results are shown in FIGS. 2 to 4, respectively. As can be seen, the minimum amount of 8a type viral DNA detected was 17.2pg, the minimum amount of 8b type viral DNA detected was 8.6pg, and the minimum amount of 11 type viral DNA detected was 10.5 pg.
Example 4 reproducibility of multiplex PCR
The multiplex PCR method was used to amplify the disease positive for type 8a, type 8b and type 11 viruses three times each, and the results are shown in FIGS. 5-7. As can be seen, the target bands can be amplified by three times of repetition, and the brightness of the bands is basically consistent.
Example 5 kit for identifying common serotype chicken inclusion body hepatitis infection
The kit for identifying the common serotype inclusion body hepatitis infection comprises the following components by using 100 dosimeters:
(1) 4800. mu.L of PCR reaction solution, which specifically included:
(2) positive plasmid: the mixture of the recombinant plasmids T-8a, T-8b and T-11 is 200 mu L, and the final concentration of each recombinant plasmid is 0.1 ng/mu L;
the detailed preparation method of the recombinant plasmids T-8a, T-8b and T-11 is as follows:
FAV-8a, FAV-8b and FAV11 virus nucleic acids were extracted according to the instructions of a virus nucleic acid extraction kit (product of AXYGEN), and target bands were amplified by PC R using the primers FAV8a-1/FAV8a-2, FAV8b-1/FAV8b-2 and FAV11-1/FAV11-2, respectively, under the following reaction conditions:
PCR reaction (50. mu.L): ddH2O38.5. mu.L, 10 XPCR buffer 5. mu.L, 2.5mM dNTP 3. mu.L, 5U/. mu.L Taq DNA polymerase 0.5. mu.L, 20 pmol/. mu.L upstream and downstream primers 0.5. mu.L, sample nucleic acid 2. mu.L; placing the mixture in a PCR amplification instrument for reaction, wherein the reaction program comprises the following steps: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 15sec, and elongation at 72 ℃ for 30 sec; extending for 5min at 72 ℃;
and recovering a target band, connecting the target band with a vector pMD18-T, transforming DH5 alpha competent cells, extracting plasmids, and sending the plasmids to Shanghai Yingjun biotechnology limited company for sequencing after positive PCR identification. The plasmid markers with correct sequences are T-8a, T-8b and T-11 and are respectively used as positive controls of FAV-8a, FAV-8b and FAV11 viruses;
the concentrations of recombinant plasmids T-8a, T-8b and T-11 were determined, respectively, and were diluted to 0.3 ng/. mu.L, mixed in equal amounts, and the final concentrations were all 0.1 ng/. mu.L.
Example 6 specific procedures for identifying common serotype chicken inclusion body hepatitis infection by kit
The method for identifying the common serotype chicken inclusion body hepatitis infection described in this embodiment is performed by using the kit described in embodiment 5, and the specific operation steps are as follows:
(1) extracting nucleic acid of a sample: taking 200 μ L of homogenate of the disease material to be detected, extracting virus nucleic acid according to the instruction of a virus nucleic acid extraction kit (product of AXYGEN company) for later use;
(2) and (3) PCR reaction: taking 48 mu L of PCR reaction solution in the kit, adding 2 mu L of sample nucleic acid, mixing uniformly, and reacting according to the following procedures: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 15sec, and elongation at 72 ℃ for 30 sec; extending for 5min at 72 ℃;
(3) positive control: taking 48 mu L of PCR reaction solution in the kit, adding 2 mu L of positive plasmid, mixing uniformly, and reacting according to the following procedures: pre-denaturation at 95 ℃ for 5 min; 30 cycles of denaturation at 94 ℃ for 15sec, annealing at 55 ℃ for 15sec, and elongation at 72 ℃ for 30 sec; extending for 5min at 72 ℃;
(4) and (3) electrophoretic observation: adding 5 mu L of the PCR product into 1.5% agarose gel for electrophoresis observation, and judging the FAV-8a virus to be positive if a band with the size of 292bp appears; if a band with the size of 208bp appears, the FAV-8b virus is judged to be positive; if a strip with the size of 474bp appears, the FAV-11 virus is judged to be positive; if the bands with the sizes of 292bp and 208bp appear, the virus can be judged to be the mixed infection of FAV-8a and FAV-8b viruses; if the bands with the sizes of 292bp and 474bp appear, the mixed infection of FAV-8a and FAV-11 viruses can be judged; if the bands with the sizes of 208bp and 474bp appear, the virus can be judged to be the mixed infection of FAV-8b and FAV-11 viruses; if the bands with the sizes of 292bp, 208bp and 474bp appear, the mixed infection of FAV-8a, FAV-8b and FAV-11 viruses can be judged.
Example 7 clinical applications
The sample for clinical application test in the embodiment is 50 samples which are sent and detected in different chicken farms in different periods. The kit and the virus separation and identification method are used for simultaneously detecting the samples to be detected, and the coincidence rate conditions of the two methods are transversely compared.
The results show that 4 parts of the 8 a-type virus positive disease material were detected by the kit according to the procedure in example 6, and the positive detection rate was 8%, whereas 4 parts of the positive disease material were detected by the virus isolation method, and the positive detection rate was 8%, and the coincidence rate of the two methods was 100% (specifically, as shown in table 1 below).
TABLE 1 comparison of the kit with the Virus isolation identification (FAV-8a)
Note: the detected coincidence rate is (common positive number + common negative number)/total sample number multiplied by 100%
The result shows that the kit detects 7 parts of 8b type virus positive disease material, the positive detection rate is 14%, the virus separation identification detects 6 parts of positive disease material, the positive detection rate is 12%, and the coincidence rate of the two methods is 98% (specifically shown in the following table 2).
TABLE 2 comparison of the kit with the Virus isolation identification (FAV-8b)
Note: the detected coincidence rate is (common positive number + common negative number)/total sample number multiplied by 100%
The result shows that the kit detects 4 parts of type 11 virus positive disease material, the positive detection rate is 8%, the virus separation and identification detects 3 parts of positive disease material, the positive detection rate is 6%, and the coincidence rate of the two methods is 98% (as shown in the following table 3).
TABLE 3 comparison of the kit with the identification of the Virus isolation (FAV-11)
Note: the detected coincidence rate is (common positive number + common negative number)/total sample number multiplied by 100%
It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications therefrom are within the scope of the invention.
SEQUENCE LISTING
<110> poultry institute of academy of agricultural sciences of Shandong province
<120> kit for identifying common serotype chicken inclusion body hepatitis infection and application thereof
<130> 2020.10.28
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<170> PatentIn version 3.5
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Claims (3)
1. A kit for identifying common serotype chicken inclusion body hepatitis infection is characterized by comprising the following components in 100 dosage:
PCR reaction system 4800. mu.L:
positive plasmid 200. mu.L;
the PCR reaction solution system comprises: detection primers FAV8a-1 and FAV8a-2 for detecting the avian adenovirus group I of serum 8a type, wherein the amplified fragment size is 292 bp; detection primers FAV8b-1 and FAV8b-2 for detecting the serum 8b type I group avian adenovirus, wherein the amplified fragment size is 208 bp; detection primers FAV11-1 and FAV11-2 for detecting the avian adenovirus group I of serotype 11, wherein the amplified fragment size is 474 bp;
the detection primers FAV8a-1 and FAV8a-2 have the following sequence structures:
FAV8a-1：5'-AACCCCTATGAGAATACCACT-3'；
FAV8a-2：5'-CCAAATATTTCGTGCTCCC-3'；
the detection primers FAV8b-1 and FAV8b-2 have the following sequence structures:
FAV8b-1：5'-GCCAGCGTTTCAGGCTCT-3'；
FAV8b-2：5'-CGTAGTAAGGCGTTGTTCCA-3'；
the detection primers FAV11-1 and FAV11-2 respectively have the following sequence structures:
FAV11-1：5'-GAAGACTTTAGCGCCTCG-3'；
FAV11-2：5'-CTCGGTTCCCTTATCACCC-3'；
the positive plasmid is a mixture of recombinant plasmids T-8a, T-8b and T-11 with the final concentration of 0.1 ng/mu L;
the recombinant plasmids T-8a, T-8b and T-11 were prepared as follows: FAV-8a, FAV-8b and FAV11 virus nucleic acids are respectively extracted, primers FAV8a-1 and FAV8a-2, FAV8b-1 and FAV8b-2, FAV11-1 and FAV11-2 are respectively used for carrying out positive PCR amplification on a target strip, the target strip is recovered and is connected with a vector pMD18-T, the target strip is transformed into DH5 alpha competent cells, plasmids are extracted, and sequencing is carried out after positive PCR identification; plasmids with correct sequences were labeled as T-8a, T-8b and T-11 and served as positive controls for FAV-8a, FAV-8b and FAV11 viruses, respectively.
2. The kit for identifying the common serotype chicken inclusion body hepatitis infection according to claim 1, wherein a PCR reaction solution system comprises the following components:
3. the kit for identifying a common serotype chicken inclusion body hepatitis infection according to claim 1 or 2, characterized in that the positive PCR amplification step reaction system (50 μ L) comprises the following components:
ddH2o38.5. mu.L, 10 XPCR buffer 5. mu.L, 2.5mM dNTP 3. mu.L, 5U/. mu.L Taq DNA polymerase 0.5. mu.L, 20 pmol/. mu.L upstream and downstream primers 0.5. mu.L, sample nucleic acid 2. mu.L.
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