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Multiplex PCR (polymerase chain reaction) detection primers for avian leukosis viruses and application thereof

A technique for detection of avian leukosis virus and primers, which is applied in the field of multiplex PCR detection primers for avian leukosis virus, can solve the problems of complicated operation, high detection cost, poor sensitivity, etc., achieve low cost, high sensitivity and specificity, and increase experimental cost Effect

Inactive Publication Date: 2012-05-02
GANSU AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above-mentioned various diagnostic methods have their own shortcomings, such as complicated operation, high detection cost, poor sensitivity, etc.

Method used

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  • Multiplex PCR (polymerase chain reaction) detection primers for avian leukosis viruses and application thereof
  • Multiplex PCR (polymerase chain reaction) detection primers for avian leukosis viruses and application thereof
  • Multiplex PCR (polymerase chain reaction) detection primers for avian leukosis viruses and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 (product embodiment)

[0019] The multiple PCR detection primer of a kind of avian leukosis virus that the present invention proposes comprises the following 3 pairs of nucleotide sequences of 3 subgroups A, B, J of avian leukosis virus:

[0020] Primers for detection of avian leukosis virus subgroup A:

[0021] Upstream primer A3: 5'-GGTTGGTCTAGACAGGAAGC-3'

[0022] Downstream primer A4: 5'-CATTGCCACAGCGGTAC-3'

[0023] Avian leukosis virus subgroup B detection primers:

[0024] Upstream primer B3: 5'-CATACGATAGTCCGGCTG-3'

[0025] Downstream primer B4: 5'-CCCCACACATCCTGACA-3'

[0026] Avian leukosis virus subgroup J detection primers:

[0027] Upstream primer J3: 5'-GGAGTTCATCTATTGCAACAACC-3'

[0028] Downstream primer J4: 5'-GCGCCTGCTACGGTGGT-3'.

[0029] Among them, the detection primers for avian leukosis virus subgroup A (ALV-A) were designed on the upstream 596-615 sites and the downstream 775-791 sites of the env gene, and the accession num...

Embodiment 2（ application Embodiment ）

[0033] The application of the multiple PCR detection primers of the avian leukosis virus proposed by the present invention in the detection of the avian leukosis virus comprises the following steps:

[0034] (1) Extract whole genome DNA from chicken blood as template for multiplex PCR;

[0035](2) Use the detection primers of avian leukosis virus subgroup A, the detection primers of avian leukosis virus subgroup B and the detection primers of avian leukosis virus subgroup J as 3 pairs of detection primers to perform multiplex PCR; the reaction conditions of the multiplex PCR are shown in Table 1 and table 2:

[0036]

[0037]

[0038] (3) Take 4 μL of the PCR amplification product from step (2) and perform 1% agarose gel electrophoresis for identification, and record the presence or absence of amplified bands, and determine the infected virus subgroup.

[0039] Through the above steps, three bands can be detected by agarose gel electrophoresis, the sizes are: 196bp, 253...

Embodiment 3

[0045] Embodiment 3 (specificity experiment)

[0046] This embodiment is an embodiment of detecting the specificity of six primers proposed by the present invention:

[0047] Separately extract: chicken Marek's disease virus (MDV)-infected cell genome, turkey herpes virus (HVT)-infected cell genome, Newcastle disease virus (NDV)-infected cell genome, infectious bursal disease virus (IBDV)-infected cell genome , Infectious laryngotracheitis virus (ILTV) infected cell genome, infectious bronchitis virus (IBV) infected cell genome, DF-1 cell genome, multiple PCR reactions were carried out by the method of Example 2, the results are as follows image 3 shown.

[0048] image 3 is the specificity test electropherogram of this method, among them, M: DNA molecular mass standard; 1: ALV-A / B / J; 2~8: control strains (MDV, HVT, NDV, IBDV, ILTV, IBV, DF -1), under multiplex PCR optimization conditions, use the genomes of MDV, HVT, NDV, IBDV, ILTV, IBV, and DF-1 cells as templates fo...

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Abstract

The invention discloses multiplex PCR (polymerase chain reaction) detection primers for avian leukosis viruses. The primers comprise detection primers for detecting subgroup A, subgroup B and subgroup J of avian leukosis viruses. The inventional also discloses a reaction system and reaction conditions of multiplex PCR. The detection method provided by the invention has the advantages of being rapid, accurate and cheap.

Description

technical field [0001] The invention relates to a method for detecting animal diseases, in particular to multiple PCR detection primers for avian leukosis virus and applications thereof. Background technique [0002] Avian leukemia is a kind of infectious disease caused by avian lekosis virus (ALV), which is mainly caused by malignant proliferation of hematopoietic cells. Includes lymphocytic leukemia, erythroblastic leukemia, myeloblastic leukemia, and myeloid leukemia. Avian leukemia mainly causes clinical and subclinical infections through vertical and horizontal transmission, resulting in non-neoplastic syndromes manifested by changes in production indicators, such as delayed sexual maturity, decreased egg production rate, egg weight, fertilization rate, and hatchability in hens Decline, which seriously affects the production performance of chickens, and eventually produces tumors, resulting in the death of chickens. And the disease can harm offspring chickens through ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
Inventor 吴润管宏伟万学瑞张小丽刘磊贾晓蕊刁小龙罗鹏何轶群
Owner GANSU AGRI UNIV
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