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Reagent kit for detecting avian leukemia virus J sub-groups

An avian leukemia virus and kit technology, applied in the field of molecular biology, can solve the problems of ALV-J gene becoming faster, more difficult to detect, unsuitable for detection, etc., and achieve the effects of improving detection efficiency, convenient operation and high specificity

Active Publication Date: 2017-02-15
CHINA AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ALV-J grows well on chicken embryo fibroblasts (CEFs), but fails to replicate or transform in mammalian cell culture
The blood, cloacal swabs, embryos or feather marrow materials of diseased chickens were treated and inoculated on CEF for culture. ALV-J could proliferate on CEF, but did not produce cytopathic changes. The isolated virus could be compared with anti-ALV-J Antiserum neutralizes the reaction, but this method needs to prepare CEF against endogenous diseases in the laboratory, which is not suitable for the detection of large quantities of samples, and the ALV-J gene becomes faster, and its antigenicity is constantly changing, so it needs to be screened and matched ALV-J-specific serum for neutralization test, which increases the difficulty of detection

Method used

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  • Reagent kit for detecting avian leukemia virus J sub-groups
  • Reagent kit for detecting avian leukemia virus J sub-groups
  • Reagent kit for detecting avian leukemia virus J sub-groups

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Embodiment 1 is used to detect the design of the specific primer of J subgroup of avian leukosis virus

[0038] The genome sequence of HLJ13SH01 (GenBank accession number: KM376510) in the NCBI database was used as a reference to design specific primers. Among the many alternative primers (shown in Table 1), after repeated screening and comparison tests to exclude possible non-specific matches between the primers and other species sequences, after repeated screening and verification, the optimized primer pair was finally obtained , each primer amplification electrophoresis results are as follows figure 1 shown. Select the primer pair 1 with the brightest amplification target band and no non-specific bands as the best primers. The upstream primer sequence is located at the end of the pol gene, and the downstream primer is located at the end of the gp85 gene to amplify a sequence with a length of about 1091bp. The primers are between 5296nt-5315nt, and the downstream pr...

Embodiment 2

[0042] Example 2 Detecting the RT-PCR Detection Method of J Subgroup Avian Leukosis Virus Optimum Annealing Temperature Exploration

[0043] 1. Pretreatment of test samples

[0044] (1) Tissue sample processing: take 100 mg of organ tissue samples, add 0.5 ml of sterilized saline, grind and suspend with a grinder, centrifuge the tissue suspension at 3000 rpm for 30 min, and take the supernatant for detection and analysis.

[0045] (2) Treatment of cloacal or oropharyngeal swab samples: add 0.5 ml sterile saline to the swab sample and oscillate to suspend with a vortex shaker, centrifuge the sample suspension at 3000 rpm for 30 min, and take the supernatant for detection.

[0046] 2. Extraction of total RNA from samples

[0047] Extraction was performed according to the instructions of Trizol RNA Extraction Kit (Invitrogen).

[0048] 3. Reverse transcription into cDNA

[0049] Add the following components into a 0.2ml centrifuge tube: 4 μl of RNA solution, 1 μl of random pri...

Embodiment 3

[0057] Example 3 Detecting the optimal cycle number of the RT-PCR detection method for J subgroup avian leukosis virus

[0058] For the pretreatment of the test sample, the extraction of the total RNA of the sample, and the reverse transcription into cDNA, refer to the corresponding method in Example 2. Using the conditions determined in Examples 1 and 2, 6 different cycle numbers (28, 29, 30, 31, 32 and 33) were designed to perform RT-PCR on ALV-J.

[0059] Add ingredients in 0.2ml centrifuge tube see Example 2. After mixing gently, use the following reaction program: initial denaturation at 94°C for 5 min, 30 cycles (94°C for 45 s, 55°C for 45 s, 72°C for 70 s), and finally extension at 72°C for 10 min. After the PCR reaction, use 1× Prepare 1% agarose gel with TAE electrophoresis buffer and mix the fluorescent dye Gelsafe according to the reference ratio, take 7 μl of PCR product and add it to the gel well, select the appropriate voltage (4V / cm-10V / cm) for electrophoresis,...

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Abstract

The invention provides a reagent kit for detecting avian leukemia virus J sub-groups (ALV-J), and belongs to the technical field of RT-PCR (reverse transcription-polymerase chain reaction) detection. The reagent kit contains a pair of specific primers. Nucleotide sequences of the specific primers are respectively shown as SEQ ID NO.1-2. The invention further provides a method for detecting the avian leukemia virus J sub-groups. The reagent kit and the method have the advantages of high specificity, sensitivity and efficiency, good universality and low cost. Besides, quick differential diagnosis can be carried out on clinical disease samples in 6.5 h, technical means can be provided for early quick diagnosis on the ALV-J and conduction of molecular epidemiological investigation, and accordingly avian leukemia prevention and control can be effectively guided during poultry raising production.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a kit for detecting the J subgroup of avian leukosis virus and its application. Background technique [0002] Avian Leukosis (AL) is a general term for a class of neoplastic diseases mainly caused by malignant proliferation of hematopoietic cells caused by Avian Leukosis virus (ALV). ALVs were divided into 10 subgroups from A to J according to the range of hosts, differences in viral envelope antigens, virus interference tests and genome molecular biological characteristics. Avian Leukosis Virus Subgroup J (ALV-J) is a new type of avian leukosis virus isolated from broiler chickens for the first time in 1991, and its prototype strain is HPRS103. ALV-J mainly causes myeloid neoplasia (ML) and various other malignant tumors in broilers. Almost all types of chickens are susceptible to ALV-J. In recent years, ALV-J infection is the main infection in my country, accounting for 65-7...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/701
Inventor 张国中冯金玲徐美玉
Owner CHINA AGRI UNIV
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