Universal primer pair for detecting pigeon type 1 adenovirus, pigeon type 2 adenovirus, duck type 3 adenovirus and avian type 4 adenovirus and kit thereof

Abstract

The invention relates to a universal primer pair for detecting pigeon type 1 adenovirus, pigeon type 2 adenovirus, duck type 3 adenovirus and avian type 4 adenovirus and a kit thereof. The sequence of the primer pair is as shown in SEQ ID NO.1-SEQ ID NO.2. The primer pair can effectively amplify four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4) at the same time, the size is about 440bp, and the primer pair can be used for detecting the four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4). The four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4) can be creatively amplified by only using two primers, if the four pathogens need to be differentially diagnosed subsequently, only the PCR amplification result needs to be subjected to clone sequencing and then BLAST comparative analysis, and the defects of a common method can be effectively avoided.

Description

Technical Field

[0001] The invention belongs to the field of poultry disease, and in particular relates to a primer pair and a kit for detecting universal pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4. Background Art

[0002] Adenovirus is a spherical structure without an envelope. Its virus particles are often arranged in a lattice in the infected cell nucleus. Each virus particle contains a 36kb linear double-stranded DNA with an inverted terminal repeat (ITR) of 100 to 600 bp at each end. The inner side of the ITR is the virus packaging signal, which is the cis-acting element required for virus packaging. The genome contains the early-expressed E1 to E4 genes related to adenovirus replication and the late-expressed L1 to L5 genes related to adenovirus particle assembly. The linear double-stranded DNA and core protein form a medullary core with a diameter of 60 to 65nm, which is wrapped in the capsid. The capsid is icosahedral symmetrical and consists of 252 capsomers with a diameter of 8 to 10nm. The capsomers are arranged on triangular faces, with 6 on each side, of which 240 are hexons (non-vertex capsomers) and the other 12 are penton bases (vertex capsomers). Each hexon is a homotrimer of hexon protein. The trimer hexon molecule has a triangular spire and a pentahedral base. The spire region is composed of four loops, namely loop1, loop2, loop3, and loop4, and the base contains two regions, P1 and P2. The epitope on the hexon is the standard for diagnosing different serotypes. It includes antigenic components of mammalian adenovirus and is the most sensitive part of the virus body to immune selection pressure. Each penton base is bound to one (mammalian adenovirus) or two (avian adenovirus) 9-77.5nm long fiber protrusions. These fibers extend from the capsid surface with penton protein as the base. The top of the fiber forms the head region. The fiber protrusions are serum-specific and contain species-specific antigenic determinants responsible for in vitro hemagglutination.

[0003] Avian adenovirus belongs to the genus Aviadenovirus of the family Adenoviridae. It is a common pathogen that infects poultry and wild birds. It generally only causes mild symptoms. The more pathogenic fowl adenovirus type 4 (FAdV-4) can cause chicken inclusion body hepatitis (IBH), gizzard erosion (GE) and pericardial effusion syndrome (HPS). Pigeon adenovirus disease is an acute infectious disease caused by pigeon adenovirus type 1 (PiAdV-1) and pigeon adenovirus type 2 (PiAdV-2) in pigeons worldwide. The main characteristics of the diseased pigeons are vomiting, dehydration and discharge of yellow thin feces. In recent years, with the rapid development of the pigeon industry, especially the racing pigeon industry, there have been many reports of cross-species infection of poultry-derived (chicken or duck-derived) diseases to pigeons, such as H5Nx subtype avian influenza virus and H9N2 subtype avian influenza virus. Among them, adenovirus has the phenomenon of cross-species barrier infection between humans and primates, bats, cats, pigs, dogs, sheep and goats. The above studies show that adenovirus has spread across host species. The present invention has carried out a reserve study on universal primer pairs and kits for detecting pigeon adenovirus type 1 (PiAdV-1), pigeon adenovirus type 2 (PiAdV-2), duck adenovirus type 3 (DAdV-3) and avian adenovirus type 4 (FAdV-4), providing scientific and technological support for the subsequent cross-species transmission of epidemic diseases and scientific prevention and control of related epidemic diseases. Summary of the invention

[0004] The object of the present invention is to provide a universal primer pair and a kit for detecting pigeon adenovirus type 1 (PiAdV-1), pigeon adenovirus type 2 (PiAdV-2), duck adenovirus type 3 (DAdV-3) and avian adenovirus type 4 (FAdV-4), which can accurately and quickly determine whether the clinical specimens contain relevant pathogens.

[0005] The purpose of the present invention is achieved through the following technical solutions:

[0006] A universal primer pair for detecting pigeon adenovirus type 1 (PiAdV-1), pigeon adenovirus type 2 (PiAdV-2), duck adenovirus type 3 (DAdV-3) and avian adenovirus type 4 (FAdV-4), the primer pair comprising:

[0007] P1P2D3F4-F: 5'-TGGCARACRGCWATGGGCATGTGG-3' (SEQ ID NO. 1);

[0008] P1P2D3F4-R: 5'-CABARGTTGCAHGGACAGT-3' (SEQ ID NO. 2).

[0009] This primer pair can effectively amplify four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4) at the same time, with a size of approximately 440 bp, but they cannot be effectively distinguished by the naked eye using conventional agarose electrophoresis.

[0010] The primer pair is used in preparing a kit for simultaneously detecting pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4.

[0011] A kit for simultaneously detecting pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4, the kit comprising the primer pair.

[0012] The method for detecting pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4 using the kit includes the following:

[0013] (1) Provide positive and negative controls for four pathogens (PiAdV-1, PiAdV-2, DAdV-3, and FAdV-4);

[0014] (2) Provide universal primer pairs:

[0015] P1P2D3F4-F: 5'-TGGCARACRGCWATGGGCATGTGG-3';

[0016] P1P2D3F4-R: 5'-CABARGTTGCAHGGACAGT-3'.

[0017] (3) Nucleic acid DNA extraction: After grinding the sample to be tested, freeze and thaw three times, centrifuge at 3000 rpm for 20 min, and then take 200 μL of the supernatant and extract it according to the viral nucleic acid extraction kit ( Viral DNA / RNA Kit) instructions for extracting nucleic acid DNA.

[0018] (4) PCR reaction: PCR amplification was performed using a PCR amplification kit ( PCR SuperMix (+dye)) was recommended for 50 μL, including 2×EasyTaq PCR SuperMix 25 μL, 1 μL of 10 μmol / L upstream / downstream primers (P1P2D3F4-F and P1P2D3F4-R), 1 μL of DNA template, and deionized water to a total volume of 50 μL. The reaction conditions were 95℃ pre-denaturation for 3 min; 95℃30s, 52℃30s, 72℃30s, 35 cycles; 72℃ extension for 7 min after the cycle.

[0019] (5) Result determination: The amplified product was subjected to electrophoresis detection. When a band of about 440 bp appeared in the sample to be tested, it was judged to be positive for infection with four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4) (Note: the specific pathogen subtype could not be determined); when there was no specific amplified band in the sample to be tested, it was judged to be negative.

[0020] (6) Determination of infection type: If the specific types of samples positive for infection with the four pathogens (PiAdV-1, PiAdV-2, DAdV-3, and FAdV-4) need to be further clarified, the PCR amplification products mentioned above can be cloned and sequenced according to conventional methods, and the clone sequencing results can be subjected to BLAST analysis (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the infection type can be determined based on the BLAST analysis results.

[0021] Compared with the prior art, the advantages of the present invention are:

[0022] Generally speaking, to detect 4 kinds of pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4), it is necessary to design specific primers respectively (2 primers are required for each, and 8 primers are required in total), and they are assembled into a detection kit after condition optimization. At this time, due to the large number of primers, problems such as mutual inhibition may occur, resulting in cumbersome or even failure of the condition optimization process. However, the present invention is creative and only uses 2 primers to amplify 4 kinds of pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4). If it is necessary to perform differential diagnosis on the 4 kinds of pathogens later, it is only necessary to clone and sequence the PCR amplification results and then perform BLAST comparison analysis. The present invention can effectively avoid the drawbacks of common methods. There are no relevant research reports at home and abroad at present, and the establishment of the present invention can fill the gaps in related fields at home and abroad.

[0023] The specific universal primers used in the present invention can effectively amplify four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4) at the same time, and can quickly determine whether the sample to be tested is infected with the four pathogens. It has good specificity and has no cross reaction to common pigeon infectious diseases (such as pigeon-derived avian influenza virus AIV, pigeon-derived avian paramyxovirus AMPV-1, pigeon-derived circovirus PTTV, pigeon circovirus PiCV, and pigeon rot virus PiRV). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 It is a schematic diagram of the results of the universal detection kit of the present invention, wherein M: DL2000 molecular weight standard; 1: PiAdV-1; 2: PiAdV-2; 3: DAdV-3; 4: FAdV-4; 5: AIV; 6: AMPV-1; 7: PTTV; 8: PiCV; 9: PiRV; 10: negative control. DETAILED DESCRIPTION

[0025] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0026] 1. Related test pathogens

[0027] The experimental pathogens pigeon adenovirus type 1 (PiAdV-1), pigeon adenovirus type 2 (PiAdV-2), duck adenovirus type 3 (DAdV-3), avian adenovirus type 4 (FAdV-4), pigeon-origin avian influenza virus (AIV), pigeon-origin avian paramyxovirus (AMPV-1), pigeon-origin circovirus (PTTV), pigeon circovirus (PiCV), and pigeon rotarovirus (PiRV) were all preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0028] 2. Primer Design

[0029] Referring to the genome sequence information of PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4, the genome sequence information was analyzed using bioinformatics software, and suitable regions of PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4 were selected as target regions for primer design, and specific primers were designed using primer design software. The sequence information is as follows:

[0030] P1P2D3F4-F: 5'-TGGCARACRGCWATGGGCATGTGG-3';

[0031] P1P2D3F4-R: 5'-CABARGTTGCAHGGACAGT-3'.

[0032] Among them, this primer pair can effectively amplify four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4) at the same time, with a size of about 440bp, but they cannot be effectively distinguished by the naked eye by conventional agarose electrophoresis.

[0033] The inventors designed multiple sets of primer pairs with reference to the genome sequence information of PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4. Experimental verification showed that only the primer pairs P1P2D3F4-F and P1P2D3F4-R could effectively amplify the four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4).

[0034] The above primers were synthesized by Sangon Biotechnology (Shanghai) Co., Ltd.

[0035] 3. Nucleic Acid Extraction

[0036] After grinding the sample to be tested, it was frozen and thawed three times, centrifuged at 3000rpm for 20min, and 200μL of the supernatant was aspirated and purified according to the viral nucleic acid extraction kit ( Viral DNA / RNA Kit) instructions for extracting nucleic acid DNA.

[0037] 4. PCR reaction

[0038] PCR amplification was performed using a PCR amplification kit ( PCR SuperMix (+dye)) was performed with the recommended 50 μL, including 2×EasyTaq PCR SuperMix 25 μL, 1 μL each of 10 μmol / L upstream / downstream primers (DPAAV-F and DPAAV-R), 1 μL of DNA template, and deionized water was added to the total volume of 50 μL.

[0039] The optimized reaction conditions were as follows: pre-denaturation at 95°C for 3 min; 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s; and extension at 72°C for 7 min after the cycle.

[0040] 5. Specificity test

[0041] Using the optimized conditions, other common pathogens in pigeons (pigeon-derived avian influenza virus AIV, pigeon-derived avian paramyxovirus AMPV-1, pigeon-derived circovirus PTTV, pigeon circovirus PiCV, pigeon rotarovirus PiRV) and negative controls were amplified, and no specific target bands were observed (see Figure 1 , schematic diagram of the results of the universal detection kit), indicating that the universal detection kit established by the present invention has good specificity.

[0042] from Figure 1It can be seen that the four pathogens (PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4) all showed a band of about 440bp. However, no specific target bands were found for other common pathogens in pigeons (pigeon-derived avian influenza virus AIV, pigeon-derived avian paramyxovirus AMPV-1, pigeon-derived circovirus PTTV, pigeon circovirus PiCV, pigeon rot virus PiRV) and negative controls.

[0043] 6. Clinical Application

[0044] 25 clinical samples of pigeons were tested for infection with PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4. The corresponding nucleic acid DNA was extracted using the EasyPure Viral DNA / RNA Kit, and PCR amplification was performed according to the optimized system and conditions. The results showed that 4 positive samples were detected, with a positive rate of 16%.

[0045] Thirty-five samples of duck samples were tested for infection with PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4. The corresponding nucleic acid DNA was extracted using the EasyPure Viral DNA / RNA Kit, and PCR amplification was performed according to the optimized system and conditions. The results showed that 6 positive samples were detected, with a positive rate of 17.14%.

[0046] 7. Accurately determine the infection type

[0047] After agarose electrophoresis of the PCR-amplified positive samples, the target bands were recovered using a gel recovery kit and cloned into a T vector. After transformation into competent cells, 8 plasmids were randomly selected for identification, and the positive recombinant plasmids were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequence determination. The obtained sequencing results were subjected to BLAST analysis (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the infection type can be determined based on the BLAST analysis results. The results show that among the 4 pigeon samples, 1 was PiAdV-1 positive and 3 were PiAdV-2 positive. Among the 6 duck samples, 5 were DAdV-3 positive and 1 was FAdV-4 positive.

[0048] To clarify the effect of the present invention, the present invention simultaneously used cells to isolate viruses from 10 positive disease samples. As a result, 8 virus strains were isolated (the virus isolation rate was 70%, and the general virus isolation required 7 days), which were positive after the method of the present invention, and the results of BLAST analysis after one-step cloning sequencing were consistent with the results of the PCR universal kit.

[0049] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A universal primer pair for detecting pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4, characterized in that: The primer pairs are: P1P2D3F4-F: 5'-TGGCARACRGCWATGGGCATGTGG-3'; P1P2D3F4-R: 5'-CABARGTTGCAHGGACAGT-3'.

2. Use of the primer pair as claimed in claim 1 in preparing a kit for simultaneously detecting pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4.

3. A kit for simultaneously detecting pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4, characterized in that: The kit comprises the primer pair as claimed in claim 1.

4. A method for detecting pigeon adenovirus type 1, pigeon adenovirus type 2, duck adenovirus type 3 and avian adenovirus type 4 simultaneously using the primer pair of claim 1, the method not being for diagnosis, characterized in that: The PCR reaction system is: The following components were contained in a 50 μL system: 2×EasyTaq PCR SuperMix 25 μL, 10 μmol / L upstream primer P1P2D3F4-F 1 μL, 10 μmol / L downstream primer P1P2D3F4-R 1 μL, DNA template 1 μL, and deionized water was added to a total volume of 50 μL; The PCR amplification conditions were as follows: pre-denaturation at 95°C for 3 min; 35 cycles of 95°C for 30 s, 52°C for 30 s, and 72°C for 30 s; and extension at 72°C for 7 min after the cycle.

5. The detection method according to claim 4, characterized in that: The results are determined as follows: the amplified product is tested by electrophoresis. When a band of about 440 bp appears in the sample to be tested, it is judged to be positive for infection with the four pathogens PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4; when there is no specific amplified band in the sample to be tested, it is judged to be negative; Then, combined with clone sequencing, the clone sequencing results are subjected to BLAST analysis, and PiAdV-1, PiAdV-2, DAdV-3 and FAdV-4 can be identified and detected at the same time based on the BLAST analysis results.

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