CN111500785A - Kit and primer group for identifying three subtypes of duck hepatitis A virus - Google Patents
Kit and primer group for identifying three subtypes of duck hepatitis A virus Download PDFInfo
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Abstract
The invention provides a kit and a primer group for identifying three subtypes of duck hepatitis A virus, belonging to the field of duck infectious disease, wherein the sequence of the primer group is shown as SEQ ID NO.1-4, the kit is constructed by utilizing the primer group and is used for identifying the three subtypes of duck hepatitis A virus, and the method is simple, convenient and quick. At present, no relevant research report of a duck hepatitis A virus universal detection kit is seen at home and abroad, and the establishment of the invention can fill the blank of relevant fields at home and abroad.
Description
Technical Field
The invention provides a kit and a primer set for identifying three subtypes (DHAV-1, DHAV-2 and DHAV-3) of duck hepatitis A virus, belonging to the field of duck pathology.
Background
In 1945, the first duck viral hepatitis Virus (DHAV-1 type duck hepatitis A Virus) is found in Changdai duck farm in New York, 1950, L evine and Fabricant firstly isolate duck viral hepatitis pathogens from chicken embryo, and then in the world, the prevalence of DHAV-1 is successively reported in many countries, the yellow average construction 792792792792 of Shanghai animal husbandry in 1963 reports that duck viral hepatitis is found in Shanghai, in 1980, Wangping et al isolate the first DHAV-1 strain in Beijing area, subsequently, the occurrence and prevalence of DHAV-1 are successively reported in Zhejiang, Fujian et al, Sujinghu et al, isolate 2 strains (B strain and G strain respectively) from diseased duck with Beijing and Guangxi suspected duck hepatitis in 1999, the duck embryo neutralization test shows that the two strains are the same serotype, but are cross immunoreactive to serotype-1, artificial infection can be found with the serotype 06liver Virus, and the DHAV-1 strain, and separate the DHAV-2 strain from Japanese duck serological infection, and the third DHAV-10 strain, and the DHAV-10 strain presents the homology between DHAV-V-10 strains, and the DHAV-V-2 strain is found in China, and the DHAV-2 strain is found in China, and the serum of the Japanese duck Virus strain is found in China, the Japanese duck Virus serotype-A Virus serotype-2 strain, the DHAV-2 strain is found in the wild duck Virus serotype-10, the DHAV-III Virus serotype-2 strain, the DHAV-III Virus of the DHAV-III Virus strain is found in the DHAV-III Virus of the DHAV-III Virus strain, the DHAV-2 strain is found in the DHAV-III Virus of the DHAV-III Virus strain, the DHAV-III Virus of the wild duck strain, the DHAV-III of the DHAV-III Virus strain of the DHAV-III, the DHAV-III Virus strain of the DH.
Genomics analysis finds that the complete genome of the duck hepatitis A virus is about 7.7kb, the single-stranded positive-strand RNA, the genomic RNA consists of a 5 ' non-coding region (UTR), an Open Reading Frame (ORF), A3 ' UTR and a poly (A) tail, the genomic RNA can be used as a genetic material for stable passage and can also be directly used as mRNA for guiding and synthesizing a complete virus polypeptide chain (polyprotein) (i.e. the open reading frame ORF) with a length of 626nt of 5 ' -UTR, and the genomic RNA contains abundant secondary structures, the length of the 3 ' -UTR of DHAV is 314-317 nt, and the DHAV is the longest virus of the 3 ' -UTR in known small RNA virus family members, and the ORF of the duck hepatitis A virus can be further cut to form L-VP 0-VP3-VP1-2A1-2A 2-2B-2C-3A-3B-3C-3D.
Aiming at the identification of three subtypes (DHAV-1, DHAV-2 and DHAV-3) of duck hepatitis A virus, generally speaking, the differential diagnosis of three pathogens needs to design specific primers for each pathogen, and then the three subtypes are further assembled into a differential diagnosis kit, so that the identification method for the three subtypes (DHAV-1, DHAV-2 and DHAV-3) of duck hepatitis A virus can be carried out simultaneously, generally speaking, 6 primers need to be designed, namely 2 primers with DHAV-1 specificity need, 2 primers with DHAV-2 specificity need, 2 primers with DHAV-3 specificity need, but the assembly is relatively difficult due to the interference between the primers when a plurality of primers are assembled into a multiplex PCR kit, and the method is not reported in related research at present. Based on practical requirements, the invention skillfully designs and optimizes a primer group by using a sequence analysis and primer design tool, only 4 primers are needed, three subtypes (DHAV-1, DHAV-2 and DHAV-3) of the duck hepatitis A virus can be amplified after condition optimization, the amplification bands are different in size, and the three subtypes (DHAV-1, DHAV-2 and DHAV-3) of the duck hepatitis A virus can be identified and diagnosed after conventional agarose gel electrophoresis analysis.
Disclosure of Invention
The invention aims to provide a kit and a primer set for identifying three subtypes of duck hepatitis A virus (DHAV-1, DHAV-2 and DHAV-3), which can accurately and quickly identify the infection conditions of the three subtypes of duck hepatitis A virus (DHAV-1, DHAV-2 and DHAV-3) in clinical detected materials.
In order to realize the technical scheme, the following technical scheme is adopted:
three subtype (DHAV-1, DHAV-2 and DHAV-3) identification primer sets of duck hepatitis A virus are disclosed, and the primers are as follows:
DHAV-F1：5’- ATTCCTGGCACATCAGAAGCA-3’；
DHAV-F2：5’- TCAACATGATGCTCAAGATGGAG-3’；
DHAV-F3：5’- GTTGAATAAGATTGAAACCATG-3’；
DHAV-R123：5’- CCASWTCCKGATTTGCCAACAAC-3’。
a duck hepatitis A virus three subtype discrimination kit comprises the primer group, can simultaneously amplify three subtypes (DHAV-1, DHAV-2 and DHAV-3) of duck hepatitis A virus and realize discrimination of the three subtypes of duck hepatitis A virus, and specifically comprises the following steps:
(1) when only an amplification band is 395bp, the duck hepatitis A virus type 1 (DHAV-1) is judged to be infected positively;
(2) when only an amplification band is 672bp, the duck hepatitis A virus type 2 (DHAV-2) infection is judged to be positive;
(3) when only an amplification band is 258bp, the duck hepatitis A virus type 3 (DHAV-3) is judged to be infected positively;
(4) when the amplified bands are 395bp and 672bp, the duck hepatitis A virus type 1 (DHAV-1) and the duck hepatitis A virus type 2 (DHAV-2) are judged to be positive in mixed infection;
(5) when the amplified bands are 395bp and 258bp, the duck hepatitis A virus type 1 (DHAV-1) and the duck hepatitis A virus type 3 (DHAV-3) are judged to be positive in mixed infection;
(6) when the amplified bands are 672bp and 258bp, the duck hepatitis A virus type 2 (DHAV-2) and duck hepatitis A virus type 3 (DHAV-3) are judged to be positive in mixed infection;
(7) when the amplified bands are 395bp, 672bp and 258bp, the duck hepatitis A virus type 1 (DHAV-1), the duck hepatitis A virus type 2 (DHAV-2) and the duck hepatitis A virus type 3 (DHAV-3) are judged to be positive in mixed infection.
The invention has the advantages that:
based on practical requirements, the invention utilizes sequence analysis and a primer design tool to optimize a primer group to obtain 4 primers, only 4 primers are needed, three subtypes (DHAV-1, DHAV-2 and DHAV-3) of the duck hepatitis A virus can be skillfully amplified after condition optimization, amplification bands are different in size, and the three subtypes (DHAV-1, DHAV-2 and DHAV-3) of the duck hepatitis A virus can be distinguished and diagnosed after conventional agarose gel electrophoresis analysis.
Drawings
FIG. 1 is a schematic diagram showing the results of the identification method for three subtypes of duck hepatitis A virus, wherein M is D L2000 molecular weight standard, DHAV-1 (lane 1), DHAV-3 (lane 2), DHAV-2 (lane 3), DHAV-1 and DHAV-3 are positive for coinfection (lane 4), DHAV-1 and DHAV-2 are positive for coinfection (lane 5), DHAV-2 and DHAV-3 are positive for coinfection (lane 6), DHAV-1, DHAV-2 and DHAV-3 are positive for coinfection (lane 7), avian influenza virus 8, avian paramyxovirus type 19, Muscovy duck reovirus 10, novel reovirus 11, avian tembusu virus 12, and negative control 13.
Detailed Description
The following examples further illustrate the invention.
Example 1
1. Relevant test pathogens
The pathogenies DHAV-1 and DHAV-3 for the test, avian influenza virus, avian paramyxovirus type 1, Muscovy duck reovirus, novel duck reovirus and avian tembusu virus are identified and stored by the animal husbandry and veterinary research institute of agricultural academy of sciences of Fujian province. Because duck type 2 hepatitis A virus (DHAV-2) is only reported in Taiwan province of China at present, related strains cannot be obtained by the team, but related strain sequences are logged in GenBank, the duck type 2 hepatitis A virus (90D strain, GenBank accession number EF 067924) is synthesized in a whole gene of Shanghai, bioengineering (Shanghai) Bingquan by using a whole gene synthesis technology.
2. Primer design
Specific primers are designed by referring to the sequence information of DHAV-1, DHAV-2 and DHAV-3 whole genes and utilizing sequence analysis and primer design tools, and the sequence information is as follows:
DHAV-F1：5’- ATTCCTGGCACATCAGAAGCA-3’；
DHAV-F2：5’- TCAACATGATGCTCAAGATGGAG-3’；
DHAV-F3：5’- GTTGAATAAGATTGAAACCATG-3’；
DHAV-R123：5’- CCASWTCCKGATTTGCCAACAAC-3’。
the primers were all synthesized by Biotechnology engineering (Shanghai) GmbH.
3. Nucleic acid extraction and cDNA preparation
Grinding a sample to be detected, repeatedly freezing and thawing for three times, centrifuging at 3000rpm for 20min, carefully sucking the corresponding obtained supernatants of DHAV-1, DHAV-3, avian influenza virus, avian paramyxovirus type 1, Muscovy duck reovirus, novel duck reovirus and avian tembusu virus 200 mu L, extracting nucleic acid RNA according to the specification of a virus nucleic acid extraction Kit (EasyPure Viral DNA/RNA Kit), carefully sucking the extracted nucleic acid RNA (DHAV-1, DHAV-3, avian influenza virus, avian paramyxovirus type 1, Muscovy duck reovirus, novel duck reovirus and avian tembusu virus) 10 mu L, and reversely transcribing the RNA into cDNA by utilizing the reverse transcription Kit (EasyScript First-Strand cDNA Synthesis Supermix).
The DHAV-2 after the whole gene synthesis can be directly subjected to PCR amplification without extracting nucleic acid RNA and preparing cDNA.
4. PCR reaction
PCR amplification is carried out by adopting 50 mu L recommended by a PCR amplification kit (2 × easy Taq PCR SuperMix (+ dye)), wherein 2 × easy Taq PCR SuperMix 25 mu L, 10 mu mol/L DHAV-F1, DHAV-F2, DHAV-F3 and DHAV-R123 are respectively 1 mu L, a template (DNA or cDNA) is 1 mu L, deionized water is added to the total volume of 50 mu L, the reaction conditions are pre-denaturation at 95 ℃ for 5min, 30 s at 95 ℃, △ T30 s at 72 ℃ for 45s for 35 cycles, and extension at 72 ℃ is carried out for 10 min after the cycle is finished, wherein the annealing temperature (△ T) is optimized between 50 ℃ and 60 ℃, and the PCR conditions of the optimized universal detection kit for the duck hepatitis A virus are that the reaction conditions are pre-denaturation at 95 ℃ for 5min, 30 s at 95 ℃, 30 s at 54.3 ℃, 30 s at 72 ℃ and 45s, 35 ℃ after the cycle is finished, the extension at 10 min after the extension at 95 ℃.
5. Determination of results
The method can simultaneously amplify three subtypes (DHAV-1, DHAV-2 and DHAV-3) of duck hepatitis A virus and can simultaneously identify the three subtypes of the duck hepatitis A virus, and the judging method comprises the following steps:
(1) when only an amplification band is about 395bp, the duck hepatitis A virus type 1 (DHAV-1) is judged to be infected positively;
(2) when only an amplification band is about 672bp, the duck hepatitis A virus type 2 (DHAV-2) is judged to be infected positively;
(3) when only an amplification band is about 258bp, the duck hepatitis A virus type 3 (DHAV-3) is judged to be infected positively;
(4) when the amplified bands are about 395bp and 672bp, the duck hepatitis A virus type 1 (DHAV-1) and the duck hepatitis A virus type 2 (DHAV-2) are judged to be positive by mixed infection;
(5) when the amplified bands are about 395bp and 258bp, the duck hepatitis A virus type 1 (DHAV-1) and duck hepatitis A virus type 3 (DHAV-3) are judged to be positive by mixed infection;
(6) when the amplified bands are about 672bp and 258bp, the duck hepatitis A virus type 2 (DHAV-2) and duck hepatitis A virus type 3 (DHAV-3) are judged to be positive by mixed infection;
(7) when the amplified bands are about 395bp, 672bp and 258bp, the duck hepatitis A virus type 1 (DHAV-1), the duck hepatitis A virus type 2 (DHAV-2) and the duck hepatitis A virus type 3 (DHAV-3) are judged to be positive in mixed infection.
6. Specificity test
Under the optimized conditions, other common pathogens (avian influenza virus, avian paramyxovirus type 1, Muscovy duck reovirus, duck novel reovirus and avian tembusu virus) of waterfowl and negative control are amplified, and no specific target band is found as a result, but corresponding target bands appear in 395bp, 672bp and 258bp of detection of three subtypes (DHAV-1, DHAV-2 and DHAV-3) of duck hepatitis A virus, so that the universal detection kit for duck hepatitis A virus, which is established by the invention, has good specificity.
7. Clinical application
The detection method comprises the steps of detecting duck hepatitis A virus infection of 44 clinical inspection duck-origin disease materials, extracting corresponding RNA by using a virus nucleic acid extraction Kit EasyPure Viral DNA/RNA Kit, and respectively carrying out reverse transcription and PCR amplification detection according to an optimized system and conditions, wherein the results show that 8 single positive duck hepatitis A virus type 1 (DHAV-1) with a positive rate of 18.18%, 0 single positive duck hepatitis A virus type 2 (DHAV-2) with a positive rate of 0, 13 single positive duck hepatitis A virus type 3 (DHAV-3) with a positive rate of 29.55%, 0 mixed infection of the duck hepatitis A virus type 1 (DHAV-1) and the duck hepatitis A virus type 2 (DHAV-2) with a positive rate of 0, 0 mixed infection of the duck hepatitis A virus type 3 (DHAV-3) and the duck hepatitis A virus type 2 (DHAV-2) with a positive rate of 0, 0 sequencing duck hepatitis A virus type 1 (DHAV-1) and the DHAV-3), 353 single positive duck hepatitis A virus type A virus (DHAV-3) with a positive rate of DHAV-type 2 (DHAV-2), and the DHAV-3, and the results show that the DHAV-3 single positive duck hepatitis A virus type 1-1, the DHAV-1 and DHAV-3, the DHAV-3 and DHAV-3 are respectively, and the DHAV-3, and DHAV-3 are obtained by the results after the clinical inspection, and the mixed duck hepatitis A virus infection of the duck hepatitis A virus, and the duck hepatitis A virus, the duck hepatitis A virus is a single positive duck hepatitis A virus, and DHAV-1-3, and DHAV-3, and the DHAV-3, and the results show that the duck hepatitis A virus sequence of the duck hepatitis A virus is a single positive duck hepatitis A virus of the duck hepatitis A virus of.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
SEQUENCE LISTING
<110> animal husbandry and veterinary institute of agricultural academy of sciences of Fujian province
<120> duck hepatitis A virus three subtype identification kit and primer group
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Claims (3)
1. The identification primer group for three subtypes of duck hepatitis A virus is characterized in that: the primer group is as follows:
DHAV-F1：5’- ATTCCTGGCACATCAGAAGCA-3’；
DHAV-F2：5’- TCAACATGATGCTCAAGATGGAG-3’；
DHAV-F3：5’- GTTGAATAAGATTGAAACCATG-3’；
DHAV-R123：5’- CCASWTCCKGATTTGCCAACAAC-3’。
2. a kit for identifying three subtypes of duck hepatitis A virus is characterized in that: comprising the primer set of claim 1.
3. The kit for identifying three subtypes of duck hepatitis A virus according to claim 2, wherein the kit identifies three subtypes of duck hepatitis A virus simultaneously:
(1) when only an amplification band is 395bp, the duck hepatitis A virus type 1 infection is judged to be positive;
(2) when only an amplification band is 672bp, judging that the duck hepatitis A virus type 2 infection is positive;
(3) when only the amplified band is 258bp, judging that the duck hepatitis A virus type 3 infection is positive;
(4) when the amplified bands are 395bp and 672bp, the duck hepatitis A virus type 1 and the duck hepatitis A virus type 2 are judged to be positive in mixed infection;
(5) when the amplified bands are 395bp and 258bp, the duck hepatitis A virus type 1 and the duck hepatitis A virus type 3 are judged to be positive in mixed infection;
(6) when the amplified bands are 672bp and 258bp, judging that the duck hepatitis A virus type 2 and duck hepatitis A virus type 3 mixed infection is positive;
(7) when the amplified bands are 395bp, 672bp and 258bp, the duck hepatitis A virus type 1, the duck hepatitis A virus type 2 and the duck hepatitis A virus type 3 are judged to be positive in mixed infection.
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| CN103088165A (en) * | 2013-01-30 | 2013-05-08 | 山东农业大学 | Multi-RT-PCR (Reverse Transcription-Polymerase Chain Reaction) method for rapidly identifying virus serotype of duck viral hepatitis |
| CN103382507A (en) * | 2013-06-08 | 2013-11-06 | 四川农业大学 | One-step dual RT-PCR detection kit of 1-type and 3-type duck hepatitis A virus, primer pairs and method thereof |
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| CN111500785B (en) | 2022-05-24 |
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