CN111500785B - 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, relevant research reports of a duck hepatitis A virus universal detection kit are not seen at home and abroad, and the establishment of the invention can fill up 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 strain of duck viral hepatitis virus (DHAV-1, duck hepatitis A virus) was found in the Changdai duck farm in New York, U.S.A. In 1950, Levine and Fabricant first isolated duck viral hepatitis pathogens from chicken embryos, and subsequently the prevalence of DHAV-1 was subsequently reported in many countries throughout the world. The discovery of duck viral hepatitis in Shanghai is reported for the first time in China, Huangjunjian of Shanghai livestock institute in 1963; in 1980, the first strain DHAV-1 was isolated by Wangping et al in Beijing. Subsequently, the occurrence and prevalence of DHAV-1 were reported in Zhejiang, Fujian, and so on. 2 viruses (respectively numbered as B strain and G strain) are separated from the killed duck suspected of duck hepatitis in Beijing and Guangxi in Sujing, equal to 5 months in 1999, duck embryo serum neutralization tests show that the two viruses are the same serotype, but the duck embryo serum neutralization tests show that the artificially infected ducklings can reproduce lesions similar to clinical cases through serological cross immune reaction with classical DHAV-1, so that the new serotype is considered to appear, the separated virus is named as novel DHAV, and genome sequence determination shows that the strain has the highest homology with Korean novel duck viral hepatitis (namely duck hepatitis A virus type 3 DHAV-3) reported by Kim. The duck hepatitis A virus isolates separated from diseased and dead pheasant ducks such as Guangdong, Shandong, Yunnan, Henan and Heilongjiang in 2010 from Yuan Yan Zhenzhen et al are subjected to serotype identification and antigen-related VPl gene sequence analysis, and the result shows that two genotypes of DHAV-1 and DHAV-3 are simultaneously prevalent in various duck groups in multiple regions in China. In addition, the research also shows that DHAV-2 is only reported in Taiwan (Tseng CH, Tsai HJ. Molecular characterization of a new serotype of duck hepatitis Virus Res, 2007, 126(1-2): 19-31.). Currently, the GenBank database contains only 2 strains of DHAV-2 genomic sequence (strain 90D, GenBank accession number EF 067924; strain 04G, GenBank accession number EF 067923). Therefore, the kit brings practical requirements for the detection kit for three serotypes of duck hepatitis A virus.
Genomic analysis shows that the complete genome of duck hepatitis A virus is about 7.7kb, and single-stranded positive-strand RNA. Genomic RNA consists of a 5 'non-coding region (UTR), an Open Reading Frame (ORF), a 3' UTR and a poly (A) tail, which is stably passaged as genetic material and can be used directly as mRNA to direct synthesis of a complete viral polypeptide chain (polyprotein), i.e., an open reading frame ORF. The 5' -UTR is 626nt long and contains abundant secondary structure. DHAV has a 3 '-UTR length of 314-317 nt, and is a virus with the longest 3' -UTR among known members of the picornaviridae family. The ORF of duck hepatitis A virus can be further cut into L-VP0-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 subtypes (DHAV-1, DHAV-2 and DHAV-3) of duck hepatitis A virus (DHAV-A) are identified by the following primers:
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 of the results of the identification method for three subtypes of duck hepatitis A virus, wherein M: DL2000 molecular weight standard; 1: DHAV-1 (lane 1); 2: DHAV-3 (lane 2); 3 DHAV-2 (lane 3): (ii) a 4: DHAV-1 and DHAV-3 were positive for coinfection (lane 4); 5: DHAV-1 and DHAV-2 were positive for coinfection (lane 5); 6: DHAV-2 and DHAV-3 were positive for coinfection (lane 6); 7: positive for co-infection of DHAV-1, DHAV-2 and DHAV-3 (lane 7); 8: avian influenza virus; 9: avian paramyxovirus type 1; 10: muscovy duck reovirus; 11: duck reovirus (reovirus); 12: avian tembusu virus; 13: and (5) negative control.
Detailed Description
The following examples further describe the invention.
Example 1
1. Pathogen of related tests
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 200 mu L of supernate of correspondingly obtained DHAV-1, DHAV-3, avian influenza virus, avian paramyxovirus type 1, Muscovy duck reovirus, novel duck reovirus and avian tembusu virus, and extracting nucleic acid RNA according to the instructions of a virus nucleic acid extraction Kit (easy pure Viral DNA/RNA Kit). Carefully absorbing 10 mu L of extracted nucleic acid RNA (DHAV-1, DHAV-3, avian influenza virus, avian paramyxovirus type 1, Muscovy duck reovirus, duck novel reovirus and avian tembusu virus), and reversely transcribing the RNA into cDNA by utilizing a 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 was performed using 50. mu.L of PCR amplification kit (2 × EasyTaq PCR Supermix (+ dye)), wherein 2 × EasyTaq PCR Supermix 25. mu.L, 10. mu. mol/L DHAV-F1, DHAV-F2, DHAV-F3 and DHAV-R123 each contained 1. mu.L of template (DNA or cDNA) 1. mu.L, and deionized water was added to a total volume of 50. mu.L. The reaction condition is pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ for 30 s, Δ T for 30 s, 72 ℃ for 45 s; after the circulation is finished, the extension is carried out for 10 min at 72 ℃. Wherein the annealing temperature (delta T) is optimized between 50 ℃ and 60 ℃, and the optimized PCR condition of the universal detection kit for duck hepatitis A virus is as follows: the reaction condition is pre-denaturation at 95 ℃ for 5 min; 35 cycles of 95 ℃ for 30 s, 54.3 ℃ for 30 s, and 72 ℃ for 45 s; after the circulation is finished, the extension is carried out for 10 min at 72 ℃.
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) infection is judged to be positive;
(3) when only an amplification band is about 258bp, the duck hepatitis A virus type 3 (DHAV-3) infection is judged to be positive;
(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 of duck hepatitis A virus infection is carried out on 44 clinical delivery detection duck-origin disease materials, corresponding RNA is extracted by using a virus nucleic acid extraction Kit EasyPure Viral DNA/RNA Kit, and reverse transcription and PCR amplification detection are respectively carried out according to an optimized system and conditions. The result shows that the duck hepatitis A virus type 1 (DHAV-1) is single positive 8 parts, and the positive rate is 18.18%; the single positive of duck hepatitis A virus type 2 (DHAV-2) is 0, and the positive rate is 0; 13 portions of duck hepatitis A virus type 3 (DHAV-3) single positive, the positive rate is 29.55%; 0 part of duck hepatitis A virus type 1 (DHAV-1) and 0 part of duck hepatitis A virus type 2 (DHAV-2) mixed infection, wherein the positive rate is 0; 0 part of duck hepatitis A virus type 3 (DHAV-3) and 0 part of duck hepatitis A virus type 2 (DHAV-2) mixed infection, wherein the positive rate is 0; duck hepatitis A virus type 1 (DHAV-1) and duck hepatitis A virus type 3 (DHAV-2) are mixed and infected by 6 parts, and the positive rate is 13.63%; the results show that the clinical samples do not have duck hepatitis A virus type 2 (DHAV-2) infection, but have single infection and mixed infection of duck hepatitis A virus type 1 (DHAV-1) and duck hepatitis A virus type 3 (DHAV-3). Performing gel recovery on the positive results, cloning and sequencing, and performing BLAST analysis on sequencing results to obtain the positive result sequences of the duck hepatitis A virus type 1 (DHAV-1) which are all duck hepatitis A virus type 1 (DHAV-1) sequences; the sequences of the positive results of the duck hepatitis A virus type 3 (DHAV-3) are all duck hepatitis A virus type 3 (DHAV-3) sequences; the sequences of the positive results of the mixed infection of the duck hepatitis A virus type 1 (DHAV-1) and the duck hepatitis A virus type 3 (DHAV-3) have corresponding nucleotide sequences of the duck hepatitis A virus type 1 (DHAV-1) and the duck hepatitis A virus type 3 (DHAV-3).
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
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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 |
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