Avian oncosis triple fluorescent PCR detection kit and detection method thereof

A detection kit and fluorescence technology, applied in the direction of microorganism-based methods, biochemical equipment and methods, microorganism measurement/inspection, etc., can solve the problems of single detection, trouble, waste of cost, etc., to achieve simple operation and avoid mutual interference , The effect of shortening the detection time

Pending Publication Date: 2018-05-29
INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, rapid detection kits for the above three viruses have been developed using fluorescent quantitative PCR technology, but due to a single detection, the detection of the three viruses is both troublesome and costly

Method used

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  • Avian oncosis triple fluorescent PCR detection kit and detection method thereof
  • Avian oncosis triple fluorescent PCR detection kit and detection method thereof
  • Avian oncosis triple fluorescent PCR detection kit and detection method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0075] Synthesis of primers and probes for triple fluorescent PCR detection of J subgroup avian leukemia, Marek and reticuloendotheliosis virus, the specific steps are as follows:

[0076] The 5'-labeled JOE fluorescent group of the J subgroup avian leukemia virus probe, the 3'-labeled fluorescent quencher BHQ1; the 5'-labeled TAMRA fluorescent group of the Marek virus probe, and the 3'-labeled fluorescent quencher BHQ1; 5'-labeled FAM fluorophore and 3'-labeled fluorescence quencher BHQ1 of the reticuloendothelial virus probe. Synthesis of primers and probes for subgroup J avian leukemia, Marek and reticuloendotheliosis viruses. Wherein, each primer and its corresponding probe are specifically:

[0077] (1) Primers and probes for J subgroup avian leukosis virus:

[0078] Upstream primer of subgroup J avian leukemia virus: 5'-GCAAGAAGGACTCTAAGA-3' (SEQ ID NO: 1)

[0079] Subgroup J avian leukosis virus downstream primer: 5'-GGTTGTTGCAATAGATGAA-3' (SEQ ID NO: 2)

[0080] J ...

Embodiment 2

[0090] The preparation of the triple fluorescent PCR detection mixture, the specific steps are as follows:

[0091] Synthesized in Example 1:

[0092] Avian leukemia subgroup J virus upstream primer: 0.4 μL / test;

[0093] Avian leukemia subgroup J virus downstream primer 0.4 μL / test;

[0094] Avian leukemia subgroup J virus probe: 0.5 μL / test;

[0095] Marek virus upstream primer: 0.6 μL / test;

[0096] Marek virus downstream primer: 0.6 μL / test;

[0097] Marek virus probe: 0.4 μL / test;

[0098] Reticuloendotheliosis virus upstream primer: 0.6 μL / test;

[0099] Reticuloendotheliosis virus downstream primer: 0.6 μL / test;

[0100] Reticuloendotheliosis virus probe: 0.6 μL / test;

[0101] qPCR MIX (purchased from Novozyme): 12.5 μL / test;

[0102] Double distilled water: 5.8μL / test;

[0103] Mix evenly to obtain a triple fluorescent PCR detection mixture for subgroup J avian leukosis, Marek and reticuloendotheliosis virus.

Embodiment 3

[0105] Preparation of Positive Control Subgroup J Subgroup Avian Leukemia, Marek and Reticuloendotheliome Virus Triple Fluorescent PCR Detection Kit:

[0106] (1) Construction of viral plasmids

[0107] 1. Construction of J subgroup avian leukosis virus plasmid: the nucleic acid of the J subgroup avian leukosis virus sample (preserved by the Institute of Animal Husbandry and Veterinary Medicine, Hubei Academy of Agricultural Sciences) was amplified by PCR method, and the nucleic acid contained in SEQ ID NO: 10 was obtained. The nucleic acid fragment of the target amplified sequence, the primer sequence is shown in SEQ ID NO: 1-2. After the amplified fragment was purified, it was cloned into the pMD18-T vector by TA, identified by sequencing, and the recombinant vector with correct sequencing result was transformed into DH5α, amplified, and the J subgroup avian leukosis virus plasmid was obtained.

[0108] 2, the construction of Marek virus plasmid: adopt PCR method to carry o...

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Abstract

The invention relates to the technical field of biological detection, and in particular, relates to a triple fluorescent PCR detection kit and detection method for detection of J subgroup avian leukosis, Marek and reticuloendotheliosis viruses. The detection kit includes a triple fluorescent PCR detection mixed liquid, a negative control and a positive control, wherein the triple fluorescent PCR detection mixed liquid contains three pairs of primers and corresponding probes thereof for amplification and detection of the J subgroup avian leukosis, Marek and reticuloendotheliosis viruses, and the nucleotide sequences are shown in SEQ ID NO:1-9. The kit and the primer probes can quickly detect the J subgroup avian leukosis, Marek and reticuloendotheliosis viruses, and have the advantages of simple operation, high sensitivity, good specificity and the like; and suspected cases can be found out and confirmed in time, and the monitoring level of three kinds of oncosis can be improved.

Description

technical field [0001] The invention relates to the technical field of biological detection, in particular to a triple fluorescent PCR detection kit for detecting J subgroup avian leukemia, Marek and reticuloendotheliosis viruses and a detection method thereof. Background technique [0002] Avian leukemia is a general term for a variety of tumor diseases in poultry caused by avian leukosis virus (ALV) and sarcoma group virus of the retroviridae family. It can lead to slow development and low egg production rate of diseased chickens. disease. The disease spreads and infects chickens in two ways, vertical and horizontal. It is an immunosuppressive disease, which causes the body's resistance to decline and immunosuppression, leading to secondary infections of other bacterial and viral diseases. In the early 1990s, a new ALV was isolated from broiler chickens, called J subgroup virus, which was presumed to be the recombination of exogenous ALV and poultry endogenous ALV genes. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12R1/93
CPCC12Q1/686C12Q1/702C12Q1/705C12Q2600/16C12Q2537/143C12Q2563/107
Inventor 罗青平汪最邵华斌温国元张蓉蓉张腾飞王红琳卢琴罗玲汪宏才赵星
Owner INST OF ANIMAL SCI & VETERINARY HUBEI ACADEMY OF AGRI SCI
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