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Multi-PCR (Polymerase Chain Reaction) primer group, kit and method for detecting A, B, J and K subgroups avian leukosis viruses

A technology of avian leukosis virus and detection kit, which is applied in the field of biotechnology detection to achieve the effects of saving time, reducing mutual interference and strong specificity

Active Publication Date: 2018-12-21
JIANGSU INST OF POULTRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

It has been reported that a multiplex PCR method for ALV-A, ALV-B and ALV-J has been established, but ALV-K, which is newly identified and widely prevalent in yellow-feathered broiler chickens and landrace chickens, has not been involved.

Method used

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  • Multi-PCR (Polymerase Chain Reaction) primer group, kit and method for detecting A, B, J and K subgroups avian leukosis viruses
  • Multi-PCR (Polymerase Chain Reaction) primer group, kit and method for detecting A, B, J and K subgroups avian leukosis viruses
  • Multi-PCR (Polymerase Chain Reaction) primer group, kit and method for detecting A, B, J and K subgroups avian leukosis viruses

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025]Embodiment 1 A kind of detection A, B, J and K subgroup multiple PCR primer set design, kit assembly and method establishment

[0026] 1. Design and synthesis of primers

[0027] Design multiple PCR primer sets based on the gene sequences of ALV-A, ALV-B, ALV-J and ALV-K published in GenBank: design a universal upstream primer PF for the pol gene sequence that is relatively conserved among the subgroups; For the gp85 gene sequence that is relatively conserved within the subgroups, but differs greatly among the subgroups, 4 downstream primers were designed respectively, ALV-A downstream primer AR, ALV-B downstream primer BR, ALV-J downstream primer JR and ALV- K downstream primer KR, the primer sequence is shown in Table 1. Primers were synthesized by Shanghai Sangon Bioengineering Co., Ltd.

[0028] SEQ ID NO.

Primer name

Primer sequence (5'→3')

Amplified fragment size (bp)

amplified virus

1

PF

GGAGAAGACACCCTTGCTG

...

Embodiment 2

[0042] Embodiment 2 A kind of multiple PCR kit specificity test that detects A, B, J and K subgroup avian leukosis virus

[0043] Use the multiplex PCR kit and method that embodiment 1 builds, extract ALV-A, ALV-B, ALV-J and ALV-K isolated strain proviral DNA, simultaneously with the H9 subtype avian influenza virus (AIV) that laboratory preserves cDNA, Newcastle disease virus (NDV) cDNA, Marek's disease virus (MDV) DNA, avian reticuloendotheliosis virus (REV) cDNA, infectious bronchitis virus (IBV) cDNA and avian reovirus (ARV) cDNA was used as a template for multiplex PCR specificity tests.

[0044] The result is as figure 2 As shown, the negative control in lane 12 has no specific amplification bands, and the positive control in lane 13 has four specific amplification bands at positions 195bp, 417bp, 567bp and 742bp, and the experiment is valid. Lanes 1 to 5 are multiple PCR amplification products of ALV-A, ALV-B, ALV-J, ALV-K proviral DNA and four subgroup ALV proviral ...

Embodiment 3

[0045] Embodiment 3 A kind of detection A, B, J and K subgroup multiplex PCR reagent kit susceptibility test

[0046] Using the multiplex PCR kit and method constructed in Example 1, after mixing the positive standard plasmids pMD19A, pMD19B, pMD19J and pMD19K, do 10-fold serial dilution, 1 × 10 9 ~1×10 0 Copy / μL; at the same time, after mixing the proviral DNA of ALV-A, ALV-B, ALV-J and ALV-K isolates, make a 10-fold serial dilution, 100ng / μL~0.1fg / μL, as a template, A multiplex PCR sensitivity test was performed.

[0047] The result is as image 3 and Figure 4 shown, for image 3 Plasmid template, lanes 1 to 10 correspond to 1×10 9 , 1×10 8 , 1×10 7 , 1 × 10 6 , 1×10 5 , 1×10 4 , 1×10 3 , 1×10 2 , 1×10 1 , 1×10 0 Copy / μL positive standard plasmid, when the concentration of plasmid template is 1×10 3 copies / μL, multiplex PCR can still amplify four specific bands of ALV-A, ALV-B, ALV-J and ALV-K; Figure 4 Proviral DNA template, lanes 1 to 10 correspond to 100...

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Abstract

The invention discloses a multi-PCR (Polymerase Chain Reaction) primer group for detecting A, B, J and K subgroups avian leukosis viruses (ALV). The multi-PCR primer group comprises a common upstreamprimer SEQ ID NO. 1 for detecting the avian leukosis virus, a downstream primer SEQ ID NO. 2 of the A subgroup avian leukosis virus, a downstream primer SEQ ID NO. 3 of the B subgroup avian leukosis virus, a downstream primer SEQ ID NO. 4 of the K subgroup avian leukosis virus and a downstream primer SEQ ID NO. 5 of the K subgroup avian leukosis virus. The invention further discloses a multi-PCR kit for detecting the A, B, J and K subgroups avian leukosis viruses; the multi-PCR kit comprises the multi-PCR primer group provided by the invention, Premix Ex Taq DNA (Deoxyribonucleic Acid) polymerase, sterile double distilled water and a negative and positive control plasmid DNA template. Furthermore, the invention further discloses a multi-PCR method for detecting the A, B, J and K subgroupsavian leukosis viruses. An experiment shows that the multi-PCR primer group provided by the invention can be used for detecting the A, B, J and K subgroups avian leukosis viruses from a sample at thesame time, has the advantages of strong specificity, high sensitivity and convenience for operation and result judgment and is suitable for detecting batch samples and primary identification of exogenous ALV.

Description

technical field [0001] The invention belongs to the field of biotechnology detection, and in particular relates to a multiplex PCR primer set, kit and method for detecting A, B, J and K subgroups of avian leukemia virus. Background technique [0002] Avian leukemia virus (ALV) is a member of the genus Alpharetrovirus in the Retroviridae family, and it can cause a variety of tumor diseases in poultry. According to the antigenic difference of viral envelope protein, virus interference experiment and host range, ALV can be divided into 11 subgroups (ALV-A~K) from A to K. There are subgroups A~E and J that naturally infect chickens. In recent years, subgroup K avian leukemia virus (ALV-K) has been isolated and identified from local breeds of chickens in my country. Among them, ALV-E is an endogenous virus, which is not or very weakly pathogenic to chickens. Except for ALV-E, the others were exogenous viruses. ALV-A and ALV-B are ubiquitous in different types of chicken flocks...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/686C12N15/11C12R1/93
CPCC12Q1/686C12Q1/702C12Q2600/16C12Q2537/143
Inventor 俞燕周生徐步高明燕姜逸程旭赵秀美吕玲韦玉勇
Owner JIANGSU INST OF POULTRY SCI
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