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Primer set and kit for detecting avian leukemia virus A/B/J/K subgroup through one-step PCR

An avian leukemia virus and primer set technology, applied in the field of molecular biology, can solve the problems of economic loss in poultry industry, lack of ALV vaccine, etc., achieve simple and rapid typing detection, reduce detection time and cost, and achieve good specificity.

Active Publication Date: 2019-02-01
吉林省畜牧兽医科学研究院
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] The prevalence and outbreak of avian leukosis in recent years have brought serious economic losses to my country's poultry industry. Currently, there is no clinically available ALV vaccine or effective drug.

Method used

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  • Primer set and kit for detecting avian leukemia virus A/B/J/K subgroup through one-step PCR
  • Primer set and kit for detecting avian leukemia virus A/B/J/K subgroup through one-step PCR
  • Primer set and kit for detecting avian leukemia virus A/B/J/K subgroup through one-step PCR

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] The design of embodiment 1 PCR primer

[0042]Download the full-length ALV-A, ALV-B, ALV-J and ALV-K gene sequences in Table 1 from the GenBank database, use DNA star software for sequence alignment, and design in the most conserved pol gene region common to the four subgroups One shared upstream primer P1, four different downstream primers P2, P3, P4 and P5 were designed in the subgroup-specific env gene region. Among them, P1 and P2 can amplify a fragment of 375 bp for detection of ALV-A, P1 and P3 can amplify a fragment of 581 bp for detection of ALV-B, and P1 and P4 can amplify a fragment of 683 bp for detection of ALV-A To detect ALV-J, a 1377 bp fragment can be amplified from P1 and P5 to detect ALV-K. Primer sequences are listed below in Table 1.

[0043] Table 1

[0044] Primer

Embodiment 2

[0045] Example 2 Standard positive plasmid construction

[0046] The specific bands amplified from ALV-A, ALV-B, ALV-J and ALV-K proviral DNA were recovered with a DNA gel extraction kit, cloned into pMD-18-T vectors, and transformed into DH5α Escherichia coli is competent, and the single clone is picked and the plasmid is extracted and sent for sequencing identification. The result is as figure 1 As shown, M: DNA molecular standard DL2000; 1: pMD-A; 2: pMD-B; 3: pMD-J; 4: pMD-K, where the plasmid for cloning the 375 bp fragment of ALV-A is named pMD-A , is the standard positive control plasmid for ALV-A subtype virus; the plasmid for cloning the 581 bp fragment of ALV-B is named pMD-B, the standard positive control plasmid for ALV-B subtype virus; the plasmid for cloning the 683 bp fragment of ALV-J is named pMD -J, the standard positive control plasmid of ALV-J subtype virus; the plasmid for cloning the 1377 bp fragment of ALV-K is named pMD-K, the standard positive contro...

Embodiment 3

[0047] Example 3 PCR method establishment and condition optimization

[0048] The extracted ALV-A, ALV-B, ALV-J and ALV-K proviral DNA were diluted to 100 ng / μl respectively, and mixed at equal concentrations to serve as templates. In a 50 μl reaction system, the primer concentration was increased from 10 pmol / L ( The upstream primer P1 is 2 μl, 4 μl, 6 μl and 8 μl respectively; the downstream primers P2, P3, P4 and P5 are each 2 μl (the results are as follows figure 2 Shown M: DNA molecular standard DL2000; 1: 2 μL of upstream primer; 2: 4 μL of upstream primer; 3: 6 μL of upstream primer; 4: 8 μL of upstream primer)), annealing temperature (53 ℃, 56 ℃, 59 ℃ (result Such as image 3 The shown M: DL 2000 standard molecular weight; 1-3: the amplification results when the annealing temperature is 53°C, 56°C, and 59°C respectively)) and other conditions were optimized, and the optimal conditions for screening and obtaining multiplex PCR reactions are shown in Table 2 below : ...

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Abstract

The present invention relates to a primer set and a kit for detecting avian leukemia virus A / B / J / K subgroup through one-step PCR, wherein the kit comprises the primer set for detecting avian leukemiavirus A / B / J / K subgroup, ALV-A, ALV-B, ALV-J and ALV-K subtype virus positive control plasmids prepared from the nucleotide sequences amplified with the primer set, and negative control, can rapidly, specifically, sensitively and efficiently detect avian leukemia and distinguish avian leukemia A / B / J / K subgroup, is suitable for epidemiological investigation of avian leukemia, and is contribute to the purification of avian leukemia.

Description

technical field [0001] The invention relates to a one-step PCR detection primer set of A / B / J / K subgroups of avian leukosis virus and a kit thereof, belonging to the technical field of molecular biology. Background technique [0002] Avian leukemia is a type of neoplastic disease mainly caused by hematopoietic cell proliferation caused by Avian leukemia virus (ALV) belonging to the genus Avian Retrovirus in the Retroviridae family. Susceptible flocks can be infected both horizontally and vertically. According to the difference of virus interference mode, host range and envelope glycoprotein, ALV was divided into 10 subgroups, A-J, and avian leukosis virus of avian origin contained 6 subgroups, A, B, C, D, E and J. Subgroup E is an endogenous avian leukosis virus without tumorigenicity. A, B, C, D, and J subgroups are exogenous avian leukosis viruses, which can induce tumors in the body, cause death and reduce production performance. C and D subgroups are relatively rare an...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/702
Inventor 曹利利宫鹏涛董航郭衍冰姚新华苑淑贤张明珠
Owner 吉林省畜牧兽医科学研究院
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