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RAA primer for detecting 12 serotypes of fowl adenovirus I group and detection method of RAA primer

An avian adenovirus and serotype technology, which is applied in the field of avian virus detection, can solve the problems of failing to achieve simultaneous detection of avian adenoviruses, unable to achieve simultaneous detection of avian adenoviruses, and singleness, and achieves low equipment requirements, high accuracy, and improved detection. The effect of efficiency

Pending Publication Date: 2021-10-29
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The third is the enzyme-linked immunosorbent assay, which can detect antigens or antibodies for accurate determination. At present, there have been many studies on the use of ELISA in the detection of avian adenoviruses.
[0005] Patent CN112746135A discloses a kind of primer probe combination based on RAA technology detection I group 4 type avian adenovirus; Patent CN107385110A discloses a kind of RPA primer for detecting poultry adenovirus serotype 4; The detection of group 4 avian adenovirus has good sensitivity and specificity, but it can only detect a single serotype, and cannot simultaneously detect 12 serotypes of avian adenovirus group I
[0006] Patent CN106868212A discloses a universal PCR detection primer for subgroup I poultry adenovirus, but it is only universal for 4 species and 6 serotypes of subgroup I poultry adenovirus A, C, D and E Verification, the simultaneous detection of 12 serotypes of avian adenovirus group I has not yet been realized

Method used

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  • RAA primer for detecting 12 serotypes of fowl adenovirus I group and detection method of RAA primer
  • RAA primer for detecting 12 serotypes of fowl adenovirus I group and detection method of RAA primer
  • RAA primer for detecting 12 serotypes of fowl adenovirus I group and detection method of RAA primer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Example 1: Optimization and establishment of RAA-LFD detection method

[0039] 1. Design of RAA primers and probes

[0040] The entire amplification process of the RAA method is carried out at room temperature and under constant temperature conditions. The design of primers and probes is the key to this method, which directly affects the effectiveness, specificity and sensitivity of the method.

[0041] In terms of primer design, the general requirements of the RAA method are: (1) primer length: 30-36 bp; (2) GC content between 40% and 60%; (3) avoid primer dimers and hairpin structures; (4) The size of the amplification product is 100-200bp, and it is recommended not to exceed 500bp.

[0042] However, the design goal of the present invention is to simultaneously detect 12 different serotypes of FadV, and the region that can be used as a target is extremely limited, which is very difficult for RAA primer design. In order to solve this difficulty, on the basis of meet...

Embodiment 2

[0058] Embodiment 2: Methodological investigation of RAA-LFD detection method

[0059] 1. Sensitivity investigation:

[0060] The DNA of 12 different serotypes of FAdV was used as a template for PCR amplification. The upstream primer was: 5'-AGGTCCTGTTCGAAGAGGATDCGCGTGGTR-3', and the downstream primer was 5'-GAHTACGTCAGCAAAAACATCGCVGCCAA-3'. The 12 fragments were connected to the pMD-18T vector to construct 12 plasmid standards carrying the gene sequence of the target site. 12 standard products that will be constructed (the copy number is 10 10 copy) was sequentially diluted to 1 copy according to a 10-fold gradient, and 10 copies were selected 7 The product of one copy to one copy was used as a sample for RAA-LFD detection, and the blank double-distilled water was used as a template as a negative control. At the same time, common PCR amplification was performed on 12 kinds of plasmid standard products with the same primers, and the sensitivity of RAA-LFD method and common PC...

Embodiment 3

[0064] Embodiment 3: clinical sample detection:

[0065] The content of FAdV-N22 in serum type 4 FAdV isolates was quantified by the Reed-Muench method, and FAdV-N22 was diluted to 10 with PBS. 4 TCID 50 / 1ml, 10 3 TCID 50 / 1ml, 10 2 TCID 50 / 1ml, 10TCID 50 / 1ml, 1TCID 50 / 1ml, 0.1TCID 50 / 1ml total 6 gradients, then the above-mentioned poisoned PBS was diluted with 6 bottles of Newcastle disease live vaccine (1000 feathers) of the same batch to obtain 10 artificially polluted respectively. 4 TCID 50 / 1000 pigeons, 10 3 TCID 50 / 1000 pigeons, 10 2 TCID 50 / 1000 pigeons, 10TCID 50 / 1000 pigeons, 1TCID 50 / 1000 feathers, 0.1TCID 50 6 bottles of live Newcastle disease vaccine (1000 parts) of FAdV per 1000 feather parts, and 1 bottle of vaccine is diluted with 1ml sterile PBS in addition, as blank control.

[0066] DNA was extracted from the above-mentioned 7 bottles of different vaccines with a virus nucleic acid extraction kit, and the virus in the artificial sim...

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Abstract

The invention discloses a RAA primer for detecting 12 serotypes of fowl adenovirus I group and a detection method of the RAA primer, and belongs to the technical field of fowl virus detection. The sequences of a primer and a probe in a RAA primer and probe combination for detecting 12 serotypes of the fowl adenovirus I group are respectively as shown in SEQ ID NO.1-SEQ ID NO.3. According to the invention, 12 different serotype FadV can be detected at the same time. In addition, only necessary reagents and isothermal amplification equipment are needed, operation is easy and convenient, and the method is particularly suitable for large-scale detection of chicken flocks in basic-level farms, so that purification of chicken flock FadV is facilitated.

Description

technical field [0001] The invention relates to the technical field of poultry virus detection, in particular to an RAA primer for detecting 12 serotypes of group I poultry adenovirus and a detection method thereof. Background technique [0002] Fowl adenovirus (Fowl adenovirus, FAdV) is a DNA virus, belonging to Adenoviridae, Adenovirus genus. FAdV is divided into three groups according to the differences in antigenicity, among which group I FAdV is the most pathogenic and causes the greatest economic loss. Group I FAdV is further divided into 5 subgroups (FAdV A-FAdV E) and 12 serotypes (FAdV-1 to 7, FAdV-8a, FAdV-8b, FAdV-9 to 11). Group I FAdV can cause diseases in chickens mainly including inclusion body hepatitis (IBH), hydropericardial syndrome (HPS) and gizzard erosion (GE). Epidemiological surveys show that the prevalence of FAdV in recent years presents the characteristics of mixed infection of multiple serotypes, among which FAdV of serotype 4 (FAdV-4), FAdV of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11
CPCC12Q1/701C12Q1/6844C12Q2600/112C12Q2521/507C12Q2522/101C12Q2531/119C12Q2565/625
Inventor 赵鹏高源王一新常爽
Owner SHANDONG AGRICULTURAL UNIVERSITY
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