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Primer set, detection method and rapid detection kit for gene detection of subgroup j avian leukosis virus

A technology for detection of avian leukosis virus and gene, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganisms, etc.

Inactive Publication Date: 2012-02-22
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the current ELISA detection kit detects group-specific antigens of ALV. Due to the existence of endogenous ALV group-specific antigens, ELISA detection has certain limitations. false positive
Another shortcoming of ELISA detection is that the avian leukemia detection kits currently used in China are all imported from abroad, and their prices are very expensive
[0008] Because of its own advantages, LAMP has been used in the detection of pathogenic microorganisms and the diagnosis of infectious diseases, such as: severe acute respiratory syndrome coronavirus (SARS- CoV), Newcastle disease virus (NDV), AIDS virus type I (HIV-1), mycobacterium detection, adenoviral conjunctivitis detection, fungal detection, etc., there is no LAMP for ALV-J gene detection. related reports

Method used

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  • Primer set, detection method and rapid detection kit for gene detection of subgroup j avian leukosis virus
  • Primer set, detection method and rapid detection kit for gene detection of subgroup j avian leukosis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0052] Internal primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0053] Internal primer BIP, the nucleotide sequence of which is shown in SEQ ID NO: 4;

[0054] Wherein, R is G or A, and Y is T or C.

[0055] Embodiment 2J Subgroup Avian Leukosis Virus Gene Rapid Detection Method

[0056] In this example, the samples selected for clinical examination were those suspected of avian leukosis infection, and the cell DNA was extracted according to the SQ Tissue DNA Kit of OMEGA Company.

[0057] The primer set designed in Example 1 was sent to Shanghai Handsome Biological Co., Ltd. for synthesis, and then the four primers were diluted to 25pM / ul respectively.

[0058] LAMP reaction system (total 25ul): 1ul each of FIP and BIP, 0.25ul each of F3 and B3, 8U of BST DNA polymerase, 2.5ul of 10× reaction buffer, 8ul of dNTP, 2.0ul of betaine, 1.0ul of magnesium sulfate, to be tested DNA sample (including negative and positive control substances) 1.0ul, repleni...

Embodiment 2J

[0059] When SYBR Green I fluorescent dye was added to the above reaction product, the color of the reaction solution changed and finally turned yellow, indicating that the sample of this example contained the gene of subgroup J of avian leukosis virus.

[0060] The betaine, large fragment of Bst DNA polymerase, magnesium sulfate, dNTP and 10× reaction buffer used in this example are all commercially available or equipped with the LAMP kit.

[0061] Embodiment 3 LAMP method detects the specificity test of J subgroup avian leukosis virus gene

[0062] In this implementation, 3 samples that have been identified as having the J subgroup avian leukosis virus gene were selected as the positive experimental group: Yangzhou Js-nt strain, Guangdong isolated sample strain SCAU-0901 and Guangdong isolated sample strain SCAU-CHN;

[0063] This embodiment selects 5 samples that have been identified and do not have the J subgroup gene of avian leukosis virus as the negative experimental gro...

Embodiment 3

[0065] The present invention carries out LAMP amplification respectively to above-mentioned positive experiment group, negative experiment group and negative and positive control, and the primer used is the primer group prepared in Example 1, and the LAMP reaction system is total system 25ul, contains internal primer FIP and BIP each 1.0ul, External primers F3 and B3 each 0.25ul, Bst DNA polymerase 1.0ul, 10× reaction buffer 2.5ul, betaine 2.0ul, magnesium sulfate 1.0ul, dNTP 8ul, DNA template 1.0ul and deionized water 7.0ul; 10× reaction buffer contains 200mM Tris-HCl pH 8.8, 100mM KCl, 100mM (NH 4 ) 2 SO 4 , 20mM MgSO 4 React with 1% Triton X-100 at a constant temperature of 63°C for 45min.

[0066] Each LAMP amplification product was equally divided into two parts, and one part was subjected to agarose gel electrophoresis, such as figure 1 As shown, the nucleic acid electrophoresis bands in the LAMP amplification results (1, 2, 3, 4) of the positive samples showed a la...

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Abstract

The invention discloses a primer set, a detection method and a rapid detection kit for gene detection of subgroup J avian leukemia virus. The primer set is composed of four primers: outer primer F3, outer primer B3, inner primer FIP and inner primer BIP. The nucleotide sequence is shown as SEQ ID NO: 1-4, wherein, R is G or A, and Y is T, U or C. The present invention adopts the above-mentioned primer set to carry out the loop-mediated isothermal amplification reaction on the DNA of the sample to be tested under the optimized optimal system, and the positive reaction shows a ladder-shaped band in the agarose gel electrophoresis, and at the same time, a chromogenic agent is added to the reaction product , a positive reaction will result in a noticeable color change. The primer set and the kit of the present invention can detect the J subgroup avian leukosis virus gene with high efficiency and high specificity, and the amplification reaction can be completed in less than 1 hour, and the detection cost is low, the time consumption is short, the yield is high, and the specificity is high. And the color difference between negative and positive results is significant, the verification rate is high, and it is more obvious and reliable.

Description

technical field [0001] The invention relates to the biological identification of J subgroup avian leukemia virus gene, in particular to a primer set, a detection method and a rapid detection kit for J subgroup avian leukemia virus gene detection. Background technique [0002] At present, there are no effective vaccines and drugs for avian leukosis at home and abroad. The main means of controlling the disease is to regularly detect avian leukosis virus (Avian Leukosis Virus, ALV) in chicken flocks, and eliminate positive chickens in time. Decontamination and elimination of ALV in chicken flocks with high degree of detection. [0003] According to the host range of the virus on chicken embryo fibroblasts of different genotypes, the interference between different viruses and the neutralization antigen characteristics of the virus envelope, ALV can be divided into A, B, C, D, E and J subgroups 6 subgroups group. Subgroups A and B are clinically common exogenous viruses, subgr...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
Inventor 曹伟胜张小桃廖明罗开健任涛
Owner SOUTH CHINA AGRI UNIV
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