Gene chip, kit and method for detecting subtype avian lymphoid leukemia virus

A lymphatic leukemia and gene chip technology, applied in the direction of biochemical equipment and methods, microbial measurement/inspection, etc., can solve the problems of time-consuming and labor-intensive detection, difficulty in wide-scale promotion and use, and inability to detect a large number of samples.

Active Publication Date: 2014-12-31
北京翎羽生物科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditionally, interference assays, host range assays with susceptible and resistant cells, or subgroup-specific antisera have been used to differentiate ALV subgroups, which are effective but time-consuming; use ELISA assays and others to identify group-specific antigens P27 The method can detect ALV, but the endogenous retrovirus expressing P27 often exists and is confused with exogenous ALV, and the ELISA detection kit based on p27 antigen has not yet been commercialized in my country, and still relies on imports, and the price Expensive and difficult to promote and use on a large scale; IFA can only detect antibodies; single-plex PCR or RT-PCR have been used to detect and identify the above viruses, but it is time-consuming and laborious to detect one by one, cannot detect a large number of samples, and cannot quickly give test results

Method used

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  • Gene chip, kit and method for detecting subtype avian lymphoid leukemia virus
  • Gene chip, kit and method for detecting subtype avian lymphoid leukemia virus
  • Gene chip, kit and method for detecting subtype avian lymphoid leukemia virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0093] Example 1. Design of probes and primers and preparation of gene chips

[0094] Step 1: Design of specific probe

[0095] From Genbank, 59 full cDNA genes of each reference strain of the three subgroups or gene sequences containing env highly variable regions were collected. A database of 59 sequences was constructed according to species, and 2 reference strain sequences for each subgroup were selected by comparison (6 in total), among which the reference strains of subgroup B were named RSV2Schmidt2Ruppin B (SRB), GenBank numbering It is AF052428, the reference strain sequence name of E subgroup is ev-1, GenBank number is AY013303, the reference strain name of J subgroup is HPRS103, and GenBank number is Z46390.

[0096] Align these six sequences with the Clustal W program of DNAStar software to find their conservative and variable regions. Using Oligo 6 software, design specific probes for each subgroup sequence database in the variant region. A total of 5 specific probes ...

Embodiment 2

[0109] Example 2. Preparation of DNA template of the sample to be tested

[0110] The J subgroup NX0101 strain was inoculated on DF-1 cells that had grown into a monolayer. The inoculated cells were cultured in DMEM maintenance medium containing 2% neonatal viviparous serum for 7 days at 37°C, and then the culture supernatant was collected and used by IDEXX The Avian Leukemia Subgroup J Antibody Detection Kit was tested by the Avian Leukemia J Subgroup Antibody Detection Kit, and then the positive cells were repeatedly frozen and thawed at -20°C for three times, centrifuged to take the supernatant, extracted cDNA with the QIAGEN DNA extraction kit, and finally tested the nucleic acid concentration. Store at -20°C for later use.

[0111] CDNA sequence of Schmidt-Ruppin B (SRB) strain and RAV-2 strain of B subgroup, cDNA sequence of E subgroup ev-1 strain and SDO5O1 strain, J subgroup HPRS-103 strain, SD07LK1 strain , SCDY1 strain and NHH strain cDNA sequence corresponding to the ta...

Embodiment 3

[0112] Example 3. Optimization of multiplex PCR amplification system

[0113] The purpose of multiplex PCR is to use as few primers as possible to amplify the target sequence, that is, the target sequence of three subgroups of strains can be amplified simultaneously in the same system, so as to reduce the experimental time and steps and improve the detection efficiency.

[0114] In this study, the gene sequence of a reference strain in each of the three subgroups was selected as the representative sequence, and the concentration was adjusted to 1ng / μL and then mixed in equal volumes. Three primers were used to simultaneously perform PCR amplification in a reaction system. , The initial primer concentration is 10μmol / L. After repeated experiments and optimization, the final reaction system and reaction conditions are shown in Table 5 and Table 6.

[0115] Table 5. Optimized multiplex PCR reaction system

[0116]

[0117]

[0118] Table 6. Optimized multiplex PCR reaction conditions

[0...

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Abstract

The invention discloses a gene chip, a kit and a method for detecting subtype avian lymphoid leukemia virus, relating to the field of animal medical diagnosis. The gene chip is characterized in that specific probe sequences of three subgroups B, E and J of avian lymphoid leukemia virus ALV are arranged on a chip substrate. The invention further provides the kit for detecting subtype avian lymphoid leukemia virus, wherein the kit further comprises three specific primers of three subgroups beside the gene chip, the three specific primers are used for amplifying the DNA of a strain to be detected through multiple PCR (Polymerase Chain Reaction), and the amplification product is hybridized with the chip to judge whether the strain to be detected is avian lymphoid leukemia virus and further judges the subtype of the strain according to the hybridization result. The chip, the kit and the detecting method provided by the invention can be used for quickly and accurately detecting a plurality kind of pathogeny in high flux, correctly diagnosing infectious diseases within a relatively short period and preventing the spread of the infectious diseases.

Description

Technical field [0001] The invention relates to the field of animal medical diagnosis, in particular to a gene chip, kit and method for detecting subgroups of typed avian leukemia virus B, E, J Background technique [0002] In recent years, chicken lymphatic leukemia (AL) has been endangering the chicken industry in our country. After chickens are infected with ALV, in addition to causing bone marrow leukemia and tumors, they can also be co-infected with avian reticuloendotheliosis virus, Marek’s virus, etc., reducing body weight gain and feed conversion rate, increasing the rate of death, and increasing the chicken The flock is susceptible to bacterial diseases, parasitic diseases, and mycoplasma diseases. Multiple infections of immunosuppressive viral diseases are common in chicken flocks in China, and most of them are recessive infections or chronic infections, which are harmful to chicken growth and immune function. Causes an impact, thus bringing important harm to the entir...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/70C12Q1/68
Inventor 杨兵张悦苏霞周宏专陈小玲徐福洲王金洛
Owner 北京翎羽生物科技有限公司
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