Primer pair and kit for detecting duck parvovirus and duck dependovirus universal type

Abstract

The present application relates to a kind of primer pair for detecting duck parvovirus and duck dependent virus universal type and its kit and detection method.The kit includes four duck parvovirus (MDPV, N-MDPV, GPV and N-GPV) and the primer pair of duck dependent virus (DAAV) universal type newly found in recent years, sequence as shown in SEQ ID NO.1-2.The specific primer used in the present application can effectively amplify four duck parvovirus (MDPV, N-MDPV, GPV and N-GPV) and the duck dependent virus (DAAV) newly found in recent years, and can quickly determine whether the above pathogen infection exists in the sample to be tested.Currently, there is no duck parvovirus and duck dependent virus (both belong to parvovirus family parvovirus subfamily dependent parvovirus genus) universal type detection kit related research report at home and abroad, and the establishment of the present application can fill the gap in related fields at home and abroad.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of poultry diseases, and particularly relates to a primer pair for detecting duck parvovirus and duck dependovirus and a detection kit thereof. BACKGROUND

[0002] According to the latest classification of the International Committee on Taxonomy of Viruses (ICTV), the Family: Parvoviridae is divided into three subfamilies: Densovirinae, Hamaparvovirinae (Chinese name to be determined) and Parvovirinae. The Parvovirinae is divided into 11 virus genera.

[0003] Goose parvovirus (GPV) and Muscovy duck parvovirus (MDPV) are the smallest single-stranded DNA viruses in animal viruses. Clinically, they are characterized by diarrhea, exudative enteritis and other symptoms, and have high morbidity and mortality, which seriously endanger the development of waterfowl breeding industry. In recent years, two new types of waterfowl parvovirus causing goose short beak and dwarf syndrome (SBDS) have appeared in major waterfowl breeding areas in China: new goose parvovirus (N-GPV) causing SBDS in Beijing ducks, semi-Muscovy ducks and tufted ducks, and recombinant duck parvovirus (N-MDPV) causing SBDS in Muscovy ducks. The above four types of duck parvovirus (MDPV, N-MDPV, GPV and N-GPV) belong to Parvovirinae Dependoparvovirus Dependoparvovirus anseriform1 (waterfowl dependovirus type 1 virus species).

[0004] In 2020, the research team found that there was a duck adenovirus infection in duck flocks in Fujian Province, and the full-length VP gene (2217bp) had a nucleotide homology of 99.5% with duck adenovirus MHH-05-2015 (GenBank accession number KX583629) in GenBank. Duck adenovirus (AAV) (also known as duck dependent virus DAAV) belongs to the Dependoparvovirus avian 2 species (Chinese name is temporarily not available) of the Dependoparvovirus genus (Dependoparvovirus) of the Parvoviridae family, and is a simple structure DNA defective virus found so far, which needs to be assisted by a helper virus (usually adenovirus) to replicate. The above research shows that there are five Parvovirinae (Parvovirus subfamily) Dependoparvovirus (Dependoparvovirus) in duck flocks: MDPV, N-MDPV, GPV, N-GPV and DAAV, which brings practical needs for clinical differential diagnosis. SUMMARY

[0005] The purpose of the present application is to provide a primer pair and a detection kit for detecting duck parvovirus and duck dependent virus universal type, which is for the five (MDPV, N-MDPV, GPV, N-GPV and DAAV) belonging to Parvovirinae (Parvovirus subfamily) in duck flocks.

[0006] The primer pair and the kit for detecting Dependoparvovirus (Dependoparvovirus) universal type can accurately and quickly determine whether the clinical sample contains related pathogens.

[0007] To achieve the above technical solutions, the following technical solutions are adopted:

[0008] A primer pair for detecting duck parvovirus and duck dependent virus universal type, the primer pair is:

[0009] DPAAV-F: 5'-CTCRGGAAATTGGCATTGCGATT-3' (SEQ ID NO. 1);

[0010] DPAAV-R: 5'-ATYCCCCARTKGTTGTTGAT-3' (SEQ ID NO. 2).

[0011] Wherein, R=A / G, Y=C / T, K=G / T.

[0012] The primer pair can simultaneously effectively amplify five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV), wherein MDPV, N-MDPV, GPV, N-GPV are 246 bp in size, and DAAV is 237 bp in size, and the two cannot be effectively distinguished by naked eye in routine agarose electrophoresis.

[0013] The duck parvovirus and duck dependovirus universal detection kit comprises the primer pair.

[0014] The use method of the kit comprises the following steps.

[0015] (1) providing positive and negative controls of five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV);

[0016] (2) providing a universal primer pair:

[0017] DPAAV-F: 5'-CTCRGGAAATTGGCATTGCGATT-3';

[0018] DPAAV-R: 5'-ATYCCCCARTKGTTGTTGAT-3'.

[0019] (3) nucleic acid DNA extraction: after grinding and treating the sample to be detected, repeatedly freeze-thawing three times, centrifuging at 3 000 rpm for 20 min, and then sucking 200 μL of supernatant, the nucleic acid DNA is extracted according to the instructions of the viral nucleic acid extraction kit (Viral DNA / RNA Kit).

[0020] (4) PCR reaction: the PCR amplification adopts the recommended 50 μL of PCR amplification kit (PCR SuperMix (+dye)), wherein 2×EasyTaq PCR SuperMix is 25 μL, 10 μmol / L upper / lower primers (DPAAV-F and DPAAV-R) are each 1 μL, DNA template is 1 μL, deionized water is 22 μL, and the total volume is 50 μL. The reaction condition is 95℃ pre-denaturation for 5 min; 95℃ for 30 s, 55℃ for 30 s, 72℃ for 30 s, 35 cycles; after the cycle, 72℃ extension for 5 min. (5) result determination: the amplification product is detected by electrophoresis, when the sample to be detected appears about 250 bp band, it is judged that the five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV) are infected positively (the specific subtype cannot be determined); when the sample to be detected has no specific amplification band, it is judged to be negative.

[0021]

[0022] ​​(6) Infection type determination: If it is necessary to further determine the specific type of the positive sample infected by the five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV), the PCR amplification product can be cloned and sequenced according to the conventional method, and the cloned and sequenced results are subjected to BLAST analysis (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the infection type can be determined according to the BLAST analysis results.

[0023] The advantages of the present application over the prior art are that:

[0024] Generally, for detection of the five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV), specific primers need to be designed (2 primers for each, a total of 10 primers), and after optimization of the conditions, a detection kit is assembled. At this time, due to the large number of primers, problems such as mutual inhibition may occur, resulting in a tedious and even failed condition optimization process. However, the present application creatively uses only two primers to amplify the five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV), and if subsequent identification and diagnosis of the five pathogens are needed, only the PCR amplification results need to be cloned and sequenced and subjected to BLAST comparison analysis, which can effectively avoid the drawbacks of the common method. Currently, there is no related research report on a universal detection kit for duck parvovirus and duck dependovirus at home and abroad, and the establishment of the present application can fill the gap in the related field at home and abroad.

[0025] The specific primer pair used in the present application can effectively amplify the five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV) at the same time, and can quickly determine whether the five pathogens (MDPV, N-MDPV, GPV, N-GPV and DAAV) are present in the sample to be detected. The specificity is good, and there is no cross reaction with common duck flock infectious diseases (avian influenza virus AIV, avian type 1 paramyxovirus AMPV-1, muscovy duck reovirus MDRV, duck new type reovirus NDRV, and avian tembusu virus ATmV). BRIEF DESCRIPTION OF DRAWINGS

[0026] Figure 1 Universal detection kit result schematic diagram, wherein M: DL2000 molecular weight marker; 1: MDPV; 2: N-MDPV; 3: GPV; 4: N-GPV; 5: DAAV; 6: AIV; 7: AMPV-1; 8: MDRV; 9: NDRV; 10: ATmV; 11: negative control.

[0027] Figure 2 Universal primer pair design schematic diagram. DETAILED DESCRIPTION

[0028] The application will be further described in connection with the following examples and drawings.

[0029] Example 1

[0030] 1. Related test pathogens

[0031] The test pathogens, i.e., Muscovy duck parvovirus (MDPV), new-type Muscovy duck parvovirus (N-MDPV), goose parvovirus (GPV) of Muscovy duck origin, new-type goose parvovirus (N-GPV) of Muscovy duck origin, duck avian dependovirus (DAAV), avian influenza virus (AIV), avian paramyxovirus type 1 (AMPV-1), Muscovy duck reovirus (MDRV), new-type duck reovirus (NDRV), and avian tembusu virus (ATmV), were preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences.

[0032] 2. Primer design

[0033] Based on the genomic sequence information of MDPV, N-MDPV, GPV, N-GPV, and DAAV, the information was analyzed by bioinformatics software, and suitable regions of MDPV, N-MDPV, GPV, N-GPV, and DAAV were selected as target regions for primer design. Specific primers were designed by using primer design software, and the sequence information is as follows:

[0034] Upstream primer DPAAV-F: 5'-CTCRGGAAATTGGCATTGCGATT-3';

[0035] Downstream primer DPAAV-R: 5'-ATYCCCCARTKGTTGTTGAT-3'.

[0036] R = A / G, Y = C / T, K = G / T.

[0037] Among them, the amplification size of MDPV, N-MDPV, GPV, and N-GPV is 246 bp, and the amplification size of DAAV is 237 bp, which cannot be effectively distinguished by naked eye under conventional agarose gel electrophoresis.

[0038] The schematic diagram of the design of the upstream primer and the downstream primer is shown in Figure 2 .

[0039] The above primers were synthesized by Shengong Bioengineering (Shanghai) Co., Ltd.

[0040] 3. Nucleic acid extraction: After grinding and processing the sample to be detected, freeze-thawing was repeated three times, and after centrifugation at 3 000 rpm for 20 min, 200 μL of supernatant was taken, and nucleic acid DNA was extracted according to the instructions of the viral nucleic acid extraction kit (Viral DNA / RNA Kit).

[0041] 4. PCR reaction​

[0042] PCR amplification was performed using a PCR amplification kit ( The reaction was performed with the recommended 50 μL of PCR SuperMix (+dye), including 25 μL of 2× EasyTaq PCR SuperMix, 1 μL each of 10 μmol / L upstream and downstream primers (DPAAV-F and DPAAV-R), 1 μL of DNA template, and 22 μL of deionized water, to a total volume of 50 μL. The reaction conditions were initial denaturation at 95°C for 5 min, followed by 35 cycles of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s, followed by extension at 72°C for 5 min.

[0043] 5. Specificity test

[0044] Using the optimized conditions, other common duck pathogens (avian influenza virus AIV, avian paramyxovirus type 1 AMPV-1, Muscovy duck reovirus MDRV, duck novel reovirus NDRV, avian Tembusu virus ATmV) and negative controls were amplified, but no specific target bands were observed (see Figure 1 , a schematic diagram of the results of the universal detection kit), indicating that the universal detection kit established by the present invention has good specificity.

[0045] 6. Clinical Application

[0046] Forty-three clinical samples of duck samples were tested for MDPV, N-MDPV, GPV, N-GPV, and DAAV infection. DNA was extracted using the EasyPure Viral DNA / RNA Kit and amplified using PCR using optimized systems and conditions. Sixteen samples were positive, for a 37.21% positive rate.

[0047] 7. Accurately determine the infection type

[0048] After agarose electrophoresis of the PCR amplification sample, the specific target band is recovered by a gel recovery kit, and then cloned into a T vector. After transformation of competent cells, 8 plasmids are randomly picked for identification. The positive recombinant plasmid is sent to Shengong Bioengineering (Shanghai) Co., Ltd. for sequence determination. The obtained sequencing results are subjected to BLAST analysis (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi), and the infection type can be determined according to the BLAST analysis results. The results show that 1 sample is positive for MDPV, 5 samples are positive for N-MDPV, 1 sample is positive for GPV, 2 samples are positive for N-GPV, 2 samples are positive for DAAV, 2 samples are positive for mixed N-MDPV and DAAV, 2 samples are positive for mixed N-GPV and DAAV, and 1 sample is positive for mixed N-MDPV, N-GPV and DAAV (no other infection types).

[0049] To clarify the effect of the present application, 11 single positive samples were subjected to virus isolation using 10-day-old duck embryos. The results show that 8 strains of viruses were isolated from the 11 single positive samples (virus isolation rate was 72.73%, and general virus isolation needs 7 days), which were positive by the method of the present application, and the BLAST analysis after one-step cloning and sequencing was consistent with the detection results of the PCR universal kit.

[0050] The above description is only the preferred embodiment of the present application, and any equivalent changes and modifications made within the scope of the present application are also included in the scope of the present application.

Claims

1. A universal primer pair for detecting duck parvovirus and duck dependent virus, characterized in that: The sequences of the primer pairs are as follows: DPAAV-F: 5'-CTCRGGAAATTGGCATTGCGATT -3'; DPAAV-R: 5'-ATYCCCCARTKGTTGTTGAT-3'; Among them, R=A / G, Y=C / T, K=G / T; The duck parvovirus is goose parvovirus GPV, Muscovy duck parvovirus MDPV, novel goose parvovirus N-GPV and genetically recombinant duck parvovirus N-MDPV.

2. A universal detection kit for duck parvovirus and duck dependent virus, characterized by: The kit comprises the universal primer pair according to claim 1; the duck parvovirus is goose parvovirus GPV, Muscovy duck parvovirus MDPV, novel goose parvovirus N-GPV and genetically recombinant duck parvovirus N-MDPV.

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