PCR detection kit for specific detection of fowl adenovirus group I and detection method thereof
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A detection kit, a technology of avian adenovirus, applied in the field of viral nucleic acid detection, can solve the problems of cumbersome, economical loss, time-consuming, etc., and achieve the effects of simple operation, good repeatability and strong specificity
Inactive Publication Date: 2017-02-22
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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At the same time, the disease can be transmitted both horizontally and vertically, which brings great challenges to the prevention and control of the disease. Therefore, the establishment of rapid, sensitive and specific diagnostic methods and kits is the key to the prevention and control of the disease
At present, the diagnostic methods for detec
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Embodiment 1
[0033] The Composition and Preparation of Group Ⅰ Poultry Adenovirus PCR Detection Kit
[0034] 1. Reagent composition
[0035] The standard strain of avian adenovirus was purchased from China Veterinary Drug Administration; 10×PCR buffer for UNG plus, HotStar Taq DNA Polymerase (5U / μL), UNG (5U / μL) and dU plus dNTP mix (12.5×) were purchased from TaKaRa Company , dU plus dNTP mix (12.5×) was diluted 12.5 times to obtain dU plus dNTP mix; primers were synthesized by Shanghai Bioengineering Company; ddH 2 O is self-made in this laboratory.
[0036] 2. Primer design and synthesis:
[0037] In GenBank, 23 strains of group I adenovirus Hexon gene complete / partial sequences were selected, covering 12 serotypes of group I adenovirus (FAdV1-12), and the sequences of these 23 strains were compared and analyzed with ClustalX software to find the most conserved The region is used as the primer design region, and the merged base Y represents C and T, K represents G and T, W represents...
Embodiment 2
[0046] The method for using the Group I poultry adenovirus PCR detection kit prepared in Example 1, the specific steps are as follows:
[0047] 1) Sample processing: Add 0.1 g of collected samples such as liver and heart to homogenate in 1 mL of PBS containing 1% double antibody for 15 min, freeze and thaw twice at -40°C / normal temperature, 6000r / min, Centrifuge for 10 minutes, take the supernatant for inspection;
[0048] 2) DNA extraction of the sample to be tested: take the supernatant of the sample to be tested processed in step 1), extract DNA, and use it as the DNA template of the sample to be tested;
[0049] 3) Prepare the PCR reaction system: take 1.0 μL each of the positive quality control product, the negative quality control product, and the DNA template of the sample to be tested obtained in step 2) and add them to different PCR reaction tubes, and add 10×PCR buffer for each tube. UNG plus 5 μL, HotStar Power Taq DNA Polymerase 0.25 μL, dU plus dNTP mix 4 μL, Gro...
Embodiment 3
[0053] Preliminary Application of PCR Detection Kit for Group Ⅰ Poultry Adenovirus
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Abstract
The invention relates to a PCR detection kit for specific detection of fowl adenovirus group I and a detection method thereof. The detection kit comprises a fowl adenovirus group I PCR reaction liquid, 10*PCR buffer for UNG plus, hot start type DNA polymerase, dU plus dNTP mix, uracil DNA glycosylase, positive quality control serum and negative quality control serum, wherein the fowl adenovirus group I PCR reaction liquid comprises an upstream primer with a concentration of 5 micrometer and the sequence is shown as the specifications as well as a downstream primer with a concentration of 5 micrometer and the sequence is shown as the specifications. When detection is conducted through the PCR detection kit, conditions for a PCR amplified reaction are that UNG treatment is conducted at a temperature of 25 DEG C for 10 min, and predegeneration is conducted at a temperature of 95 DEG C for 2 min; degeneration is conducted at a temperature of 98 DEG C for 10 s, annealing is conducted at a temperature of 55 DEG C, extension is conducted at a temperature of 72 DEG C for 1 min, and all that are conducted for 30 work cycling; extension is conducted at a temperature of 72 DEG C for 10 min. The PCR detection kit for the specific detection of the fowl adenovirus group I has the advantages of being high in sensitivity (still positive when the reaction liquid is diluted to 1:105) and good in repeatability. The PCR detection method for the specific detection of the fowl adenovirus group I is easy, convenient and fast to operate, suitable for early diagnosis of fowl adenovirus group I virus, and can meet the demand for disease prevention and control in time.
Description
technical field [0001] The invention relates to a PCR detection kit for specific detection of Group I poultry adenovirus and a detection method thereof, belonging to the field of viral nucleic acid detection. Background technique [0002] Fowl adenovirus (Fowl Adenovirus, FAdV) belongs to Adenoviridae family of avian adenovirus, according to the group-specific antigens, adenoviruses can be divided into three groups: group I, group II and group III. Group I avian adenoviruses are mainly obtained from chickens, turkeys, geese and other poultry, and are divided into five species (A-E). The viruses in each species are further divided into 12 serotypes (FAdV1- 12), they have a common group antigen. Group I poultry adenoviruses mainly cause inclusion body hepatitis and hydropericardium syndrome, with high morbidity and mortality. The disease broke out in many provinces in my country in 2015-2016, causing serious economic losses to the poultry industry. At the same time, the dise...
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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCC12Q1/701C12Q1/6848C12Q2531/113C12Q2521/531
Inventor 罗玲温国元罗青平邵华斌王红琳汪宏才卢琴张腾飞艾地云张蓉蓉李林涛
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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