Primer group for identifying and detecting two fowl adenoviruses in chicken flocks and kit thereof

Abstract

The invention relates to a primer group and a kit for identifying and detecting two fowl adenoviruses (FAdV-4 and EDSV) in chicken flocks, the sequences of the primer group are as shown in SEQ ID NO.1-3, and the kit comprises the primer group. Based on the nucleotide sequence characteristics of ORF gene conserved regions coded by genomes of the two fowl adenoviruses (FAdV-4 and EDSV) in the chicken flocks, the primer group and the kit are used for identifying and detecting the fowl adenoviruses (FAdV-4 and EDSV) in the chicken flocks. And a PCR detection method for simultaneously amplifying the FAdV-4 and the EDSV only by using three primers is established. The lowest detection limit of the virus FAdV-4 is 5.07 * 10 < 3 > pg, and the lowest detection limit of the EDSV is 5.15 * 10 < 2 > pg. The method has good specificity, sensitivity and reliability, and an economic, accurate and effective method is provided for differential diagnosis of the two fowl adenoviruses (FAdV-4 and EDSV) in chicken flocks.

Description

Technical Field

[0001] The invention belongs to the field of chicken disease infectious diseases, and particularly relates to a primer set and a kit for identifying and detecting two avian adenoviruses (FAdV-4 and EDSV) in chickens. Background Art

[0002] According to the International Committee on Taxonomy of Viruses, the Adenoviridae family comprises five genera: AT-rich adenovirus, avian adenovirus, piscine adenovirus, mammalian adenovirus, and sialidase virus. The adenovirus genome is approximately 33 kilobases (kd) long and consists of linear double-stranded DNA. The virus particle consists of a non-enveloped nucleocapsid with icosahedral symmetry. The nucleocapsid contains three main structural proteins: hexon, fiber, and penton.

[0003] Highly pathogenic avian adenovirus serotype 4 (FAdV-4) belongs to the genus Avian Adenovirus in the family Avian Adenoviridae. FAdV-4 primarily infects broilers, laying hens, and breeder chickens. Since 2015, an outbreak has occurred in my country, with mortality rates as high as 80%. In recent years, it has become prevalent in waterfowl, causing severe economic losses to the poultry industry. Egg drop syndrome virus (EDSV), a member of the genus AT-rich Adenovirus in the family Avian Adenoviridae, was first reported in 1976, hence its name, EDS-76. EDSV is an infectious agent that causes decreased egg production in breeder and commercial laying hens. This disease is characterized by decreased egg production, accompanied by soft-shelled, thin-shelled, cracked, and shellless eggs.

[0004] Currently, there are two pathogens in chickens that belong to the Adenoviridae family but belong to different adenovirus genera: FAdV-4 and EDSV. Generally speaking, two primers are required to detect one pathogen (one targeting the upstream and downstream regions of the pathogen), and four primers are required to detect two pathogens. However, too many combinations of primers may lead to nonspecific competition and affect the PCR amplification effect, affecting the accurate judgment of the results. Technicians in this field need to optimize and improve this part of the technology, and there are uncertainties. Therefore, the present invention provides a primer set (only three primers are required, and the optimization of the strips is simpler and more economical than four primers). After the conditions are optimized, it can be used to identify and detect two avian adenoviruses (FAdV-4 and EDSV) in chickens. Summary of the Invention

[0005] The purpose of the present invention is to provide a primer set and a kit for the identification and detection of two avian adenoviruses (FAdV-4 and EDSV) in chickens, to establish a method that only requires one set of primers (3 primers) to identify and detect two avian adenoviruses (FAdV-4 and EDSV) in chickens, and to provide technical support for the subsequent development of related pathogenic epidemiological agents.

[0006] The purpose of the present invention is achieved through the following technical solutions:

[0007] A primer set for the differential detection of two avian adenovirus serotypes 4, avian adenovirus and egg drop syndrome virus (FAdV-4 and EDSV) in chickens, the sequence of the primer set is shown below:

[0008] F4F0: 5'-TCAACTCGGAGCGTTCGGGCAT-3' (SEQ ID NO. 1);

[0009] EDSVF0: 5'-TGGAGTTACTCTGCCAGCTAAC-3' (SEQ ID NO. 2);

[0010] F4EVR1: 5'-ATCATGTTRGGGTCTTT-3' (SEQ ID NO. 3).

[0011] The present invention also provides a detection kit for identifying and detecting two avian adenoviruses, FAdV-4 and EDSV, in chickens, which comprises the primer set.

[0012] The PCR amplification system and amplification conditions of the kit are as follows:

[0013] PCR amplification system: Amplification was performed according to the 20 μL system recommended by the PCR amplification kit (DreamTaq Green PCR Master Mix), including 10 μL of 2× PCR Master Mix amplification reaction solution, 1.0 μL of primer F4F0 (10 μM), 1.0 μL of primer EDSVF0 (10 μM), 2.0 μL of primer F4EVR1 (10 μM), 1.0 μL each of extracted nucleic acid templates (FAdV-4 and EDSV), and sterile deionized water to a final volume of 20 μL. After mixing, PCR amplification was performed.

[0014] Amplification conditions: Amplification conditions were as follows: pre-denaturation at 95°C for 3 minutes followed by cycling: denaturation at 95°C for 30 seconds, annealing at ΔT (50°C-60°C) for 30 seconds, and extension at 72°C for 30 seconds. After 35 cycles, a final extension at 72°C for 10 minutes was performed. After completion of the reaction, the reaction was identified by conventional agarose gel electrophoresis. ΔT (50°C-60°C) indicates that the annealing temperature was optimized within this range. The optimized optimal annealing temperature was 55°C.

[0015] The detection kit simultaneously detects serotype 4 avian adenovirus and egg drop syndrome virus in chickens, and the determination method is as follows:

[0016] (1) When only the amplified band of 747 bp appears, it is judged as positive for serotype 4 fowl adenovirus FAdV-4 infection;

[0017] (2) When only the amplified band of 306 bp appeared, it was judged to be positive for EDSV infection;

[0018] (3) When two amplified bands of 747 bp and 306 bp appeared, it was judged to be positive for mixed co-infection of serotype 4 fowl adenovirus FAdV-4 and egg drop syndrome virus EDSV.

[0019] Compared with the prior art, the advantages of the present invention are:

[0020] Based on the nucleotide sequence characteristics of the conserved regions of the ORF genes encoded by the genomes of two avian adenoviruses (FAdV-4 and EDSV) in chickens, this paper establishes a PCR detection method that can simultaneously amplify FAdV-4 and EDSV with only three primers. The minimum detection limit of the virus FAdV-4 is 5.07×10 3 pg, EDSV is 5.15×10 2 The present invention has good specificity, sensitivity and reliability, and provides an economical, accurate and effective method for differential diagnosis of two avian adenoviruses (FAdV-4 and EDSV) in chickens.

[0021] The present invention is simple to optimize, uses fewer primers (low cost), lays a foundation for conducting epidemiological investigations of relevant pathogens in chickens and scientifically preventing and controlling related diseases, and has very important research significance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 It is a schematic diagram of the design principle of the F4F0 primer of the present invention.

[0023] Figure 2 Schematic diagram of the design principle of EDSVF0 primers of the present invention.

[0024] Figure 3 Schematic diagram of the design principle of F4EVR1 primer of the present invention

[0025] Figure 4 1 is an electrophoresis diagram of the PCR product of the present invention; wherein, 1 is FAdV-4; 2 is EDSV; 3 is a mixture of FAdV-4 and EDSV; 4 is MDV; 5 is ALV; 6 is CIAV; 7 is CAstV; and 8 is sterile deionized water.

[0026] Figure 5 This is the result of the FAdV-4 sensitivity test; M: DNA molecular weight standard; 1: 5.07×10 5 pg;2:5.07×10 4 pg;3:5.07×10 3 pg;4:5.07×10 2 pg;5:5.07×10 1 pg;6:5.07×10 0 pg;7:5.07×10 -1 pg;8:5.07×10 -2 pg;9-10: negative control for the experiment.

[0027] Figure 6 This is the result of EDSV sensitivity test; M: DNA molecular weight standard; 1: 5.15×10 5 pg;2:5.15×10 4 pg;3:5.15×10 3 pg;4:5.15×10 2 pg;5:5.15×10 1 pg;6:5.15×10 0 pg;7:5.15×10 -1 pg;8:5.15×10 -2 pg;9-10: negative control for the experiment. DETAILED DESCRIPTION

[0028] The present invention is described in detail below with reference to the accompanying drawings and embodiments:

[0029] 1. Materials

[0030] 1.1 Strains and strains

[0031] The experimental pathogens FAdV-4 (GDMZ strain, GenBank accession number MG856954.1) and EDSV (JL strain, GenBank accession number MT646045.1) were deposited by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences. The control strains avian leukosis virus (ALV), chicken infectious anemia virus (CIAV), chicken astrovirus (CAstV), and Marek's disease virus (MDV) were also deposited by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0032] 1.2 Primer design

[0033] Based on the genome sequences of the representative strains of FAdV-4 of the family Adenoviridae on the GenBank database of the National Center of Biotechnology Information (NCBI), including GDMZ strain (MG856954.1), HB1510 strain (KU587519.1), KR5 strain (HE608152.1), and the representative strains of EDSV, JL strain (MT646045.1), AV-127 strain (OP712666.1), and FJ12025 strain (KF286430.1), the appropriate ORF was selected. Subsequently, the primer set was designed using primer design software based on the selected ORF gene sequences. The sequences are as follows:

[0034] F4F0: 5'-TCAACTCGGAGCGTTCGGGCAT-3';

[0035] EDSVF0: 5'-TGGAGTTACTCTGCCAGCTAAC-3';

[0036] F4EVR1: 5'-ATCATGTTRGGGTCTTT-3'.

[0037] Among them, F4F0 is only targeted at the upstream region of FAdV-4 in two avian adenoviruses (FAdV-4 and EDSV) in chickens, and does not target the upstream region of EDSV (see Figure 1 ); EDSVF0 is only for the upstream region of EDSV in two avian adenoviruses (FAdV-4 and EDSV) in chickens, but not for the upstream region of FAdV-4 (see Figure 2 ); F4EVR1 is a common region of downstream primers targeting two pathogens of avian adenovirus (FAdV-4 and EDSV) in chickens (see Figure 3 The above primer sets were synthesized by Sangon Biotech (Shanghai) Co., Ltd. and produced a single positive amplification of 747 bp for FAdV-4 and 306 bp for EDSV. In chickens co-infected with two avian adenoviruses (FAdV-4 and EDSV), two bands were detected after positive amplification, with sizes of 747 bp and 306 bp, respectively.

[0038] 1.3 Main Reagents

[0039] The PCR amplification kit DreamTaq Green PCR Master Mix (2×) was purchased from Thermo Scientific, the nucleic acid genome extraction kit SimplyP Virus DNA / RNA Extraction Kit was purchased from Hangzhou Biori Technology Co., Ltd., and the reverse transcription kit Reverse Transcriptase Kit and DNA molecular weight standard DL2000 were purchased from Beijing Quanshijin Biotechnology Co., Ltd.

[0040] 2 Establishment of test method

[0041] 2.1 Viral nucleic acid extraction and reverse transcription

[0042] Genomic nucleic acids from FAdV-4, EDSV, MDV, ALV, CIAV, and CAstV were extracted using the nucleic acid genomic extraction kit. RNA from ALV, CIAV, and CAstV was reverse transcribed into cDNA using the Reverse Transcriptase Kit and stored at -80°C until use.

[0043] 2.2 Optimization of reaction solution configuration and annealing temperature

[0044] Amplification was performed according to the 20 μL system recommended by the DreamTaq Green PCR Master Mix (2×) kit, including 10 μL of 2× PCR Master Mix amplification reaction solution, 1.0 μL of primer F4F0 (10 μM), 1.0 μL of primer EDSVF0 (10 μM), 2.0 μL of primer F4EVR1 (10 μM), 1.0 μL each of the extracted nucleic acid templates (FAdV-4 and EDSV), and sterile deionized water to a final volume of 20 μL. After mixing, PCR amplification was performed.

[0045] Amplification conditions included a 3-minute pre-denaturation at 95°C followed by cycling, followed by 35 cycles of denaturation at 95°C for 30 seconds, annealing at a delta T (50°C-60°C) for 30 seconds, and extension at 72°C for 30 seconds. After 35 cycles, a final extension at 72°C for 10 minutes was performed. After completion of the reaction, the reaction was analyzed by conventional agarose gel electrophoresis. The delta T (50°C-60°C) indicates that the annealing temperature was optimized within this range. The optimized optimal annealing temperature was 55°C.

[0046] The results can be seen ( Figure 4 ), when only FAdV-4 was added as the template, a band of 747 bp appeared (lane 1); when only EDSV was added as the template, a band of 306 bp appeared (lane 2); when a mixture of FAdV-4 and EDSV was added as the template, two bands of 747 bp and 306 bp appeared (lane 3).

[0047] 2.3 Specificity test

[0048] The optimized conditions were used to amplify MDV, ALV, CIAV, CAstV and sterile deionized water as a control, but no amplified bands were observed. Figure 4 , MDV (lane 4), ALV (lane 5), CIAV (lane 6), CAstV (lane 7) and sterile deionized water (lane 8), indicating that the established method has strong specificity and has no cross-reaction to common pathogens in chickens (see Figure 4 ).

[0049] 2.4 Sensitivity test

[0050] The genomic DNA of FAdV-4 and EDSV was extracted and the OD values ​​were measured and serially diluted 10-fold. The concentration of FAdV-4 was 5.07×10 5 -5.07×10 -2 pg; EDSV concentrations were 5.15×10 5 -5.15×10 -2 The results showed that the minimum detection limit of FAdV-4 was 5.07×10 3 pg( Figure 5 ); the minimum detection limit of EDSV is 5.15×10 2 pg( Figure 6 ).

[0051] 3 Clinical Application

[0052] After 33 clinical chicken samples were ground and processed according to conventional methods, the corresponding genomic DNA was extracted using the nucleic acid genome extraction kit, and the optimized method was used to detect FAdV-4 and EDSV infection. The results showed that 3 samples were single-positive for FAdV-4, 2 samples were single-positive for EDSV, and 1 sample was mixed with FAdV-4 and EDSV. The positive target fragments after the PCR reaction were cut and recovered using an agarose gel recovery kit. According to the instructions of the pEASY-T1 SimpleCloning Kit cloning and ligation kit, the target gene fragment was cloned into the pEASY-T1 vector. After 8 single colonies were randomly picked and cultured in LB liquid medium resistant to ampicillin (content of 100μg / mL) for 14 hours, the positive recombinant plasmids screened out were sequenced. The sequencing results were verified by BLAST analysis on NCBI, and all of them were corresponding Hexon gene fragments of FAdV-4 and EDSV. Among them, the homology of 4 FAdV-4 positive fragments (including 3 single positive fragments and 1 mixed positive fragment) and the Hexon gene of FAdV-4 (GDMZ strain, GenBank accession number MG856954.1) was all above 99.0%; the homology of 3 EDSV positive fragments (including 2 single positive fragments and 1 mixed positive fragment) and the Hexon gene of EDSV (JL strain, GenBank accession number MT646045.1) was all above 99.0%, which was consistent with the experimental expectations.

[0053] The above describes an embodiment of the present invention, but this embodiment is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Ordinary technicians in this field can also make many forms based on the inspiration of this embodiment, all of which are protected by this embodiment.

Claims

1. A primer set for the differential detection of two avian adenovirus serotype 4 and egg drop syndrome virus in chickens, characterized by: The sequence of the primer set is as follows: F4F0: 5'-TCAACTCGGAGCGTTCGGGCAT-3'; EDSVF0: 5'-TGGAGTTACTCTGCCAGCTAAC-3'; F4EVR1: 5'-ATCATGTTRGGGTCTTT-3'.

2. A detection kit for identifying and detecting two avian adenovirus serotype 4 and egg drop syndrome virus in chickens, characterized by: It comprises the primer set according to claim 1.

3. The detection kit according to claim 2, characterized in that: The detection kit simultaneously detects serotype 4 avian adenovirus and egg drop syndrome virus in chickens, and the determination method is as follows: (1) When only the amplified band of 747 bp appears, it is judged as positive for serotype 4 fowl adenovirus FAdV-4 infection; (2) When only the amplified band of 306 bp appeared, it was judged to be positive for EDSV infection; (3) When two amplified bands of 747 bp and 306 bp appeared, it was judged to be positive for mixed co-infection of serotype 4 fowl adenovirus FAdV-4 and egg drop syndrome virus EDSV.

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