K subgroup avian leukosis virus detection kit

An avian leukemia virus and kit technology, applied in the field of molecular biology, can solve the problems of difficult screening, affecting primer specificity, small space, etc., to reduce costs and complicated operations, strong specificity and sensitivity, and shorten the detection period. Effect

Inactive Publication Date: 2018-05-18
SOUTH CHINA AGRI UNIV
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  • Abstract
  • Description
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Problems solved by technology

[0003] Although the fluorescent quantitative PCR detection technology is very mature, there are few reports on the fluorescent quantitative detection method for ALV-K virus, mainly because the nucleotide sequence of ALV-K is homologous to other subgroups of avian leukosis virus (ALV) The specificity is very high, which is not conducive to the selection of specific target sequences. For example, the homology between the full-length nucleotide sequence of ALV-K and the full-length nucleotide sequence of endogenous avian leukosis virus (ALV-E) is 93.7%-97.5% Between the two subgroups, there is only a significant difference in the sequence of the envelope protein gene env of about 166bp. Secondly, the homology of the gp85 gene between the foreign isolates of ALV-K and the isolates from different regions in China is 94.5%- 99.9%, which further narrows the space for selecting sequences for primer design, resulting in less space for ALV-K conservative sequences to choose from, and greater difficulty in screening. Affects the specificity of primers to a great extent, leading to inaccurate detection results

Method used

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  • K subgroup avian leukosis virus detection kit
  • K subgroup avian leukosis virus detection kit
  • K subgroup avian leukosis virus detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0036] 1. Design of fluorescent quantitative PCR primers

[0037] According to the host range of avian leukosis virus (ALV), the interference characteristics of the virus envelope, the type of cross-neutralization reaction and the molecular characteristics of the genome, the avian leukemia virus (ALV) can be divided into 11 subgroups from A to K, of which chicken-derived ALV There are 7 subgroups A, B, C, D, E, J and the new K subgroup, and the nucleotide sequences of other subgroups have different degrees of homology with the ALV-K nucleotide sequence, among which The nucleotide sequence homology between endogenous avian leukosis virus (ALV-E) and ALV-K is the highest (93.7%-97.5%), and there are only two parts of nucleotide sequences with large differences in the gp85 region (region 1: 5836-5925 is 77bp in length, region 2: 6039-6116bp is 89bp in length). In order to exclude the interference of ALV-E and other subgroups, referring to the sequences of multiple strains of ALV...

Embodiment 2

[0054] Example 2 Conventional PCR Sensitivity Experiment

[0055] The positive plasmid obtained in Example 1 was treated with dd H 2 O was serially diluted 10-fold, wherein lanes 1-8 were 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 copy / μL, lane 9 is a negative control, amplified according to the system and procedure described in Example 1, the results are as follows figure 2 As shown, the band size is 84bp;

[0056] The routine PCR positive plasmid was used ddH 2 O sequentially carry out 10-fold serial dilution, wherein 1-8 is 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 copies / μL, 9 is the negative control, amplified according to the conventional PCR system and procedures, the results are as follows image 3 As shown, the band size is 1214bp; according to figure 2 The results show that the conventional PCR reaction with fluorescent quantitative PCR primers can detect a minimum of 10 4 copies / μL.

[0057] according to image 3 As a result, it can ...

Embodiment 3

[0058] Embodiment 3 fluorescence quantitative PCR sensitivity experiment

[0059] The positive plasmid that embodiment 1 makes is used ddH 2 O sequentially carry out 10-fold serial dilution, wherein 1-8 is 10 8 、10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 copies / μL, the diluted positive plasmid standard was subjected to fluorescent quantitative PCR reaction, the 20 μL reaction system was 0.5 μL of 10 μM upstream primer, 0.5 μL of 10 μM downstream primer, 10 μL of 2×iTaq UniversalSYBR Green Supermix, 1 μL of cDNA template , RNase free H 2 O 8 μL. The reaction program was: pre-denaturation at 95°C for 3 min; 40 cycles of 95°C for 15 s and 60°C for 34 s, collecting fluorescence at 60°C for each cycle; 95°C for 15 s, 60°C for 1 min, and 95°C for 15 s. And make a kinetic curve, the result is as Figure 4 shown.

[0060] according to Figure 4 The results show that the sensitivity detection of fluorescent quantitative PCR can detect as low as 10 1 copies / μL of positive pl...

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Abstract

The invention discloses a K subgroup avian leukosis virus detection kit, which relates to the technical field of virus fluorescent quantitative PCR detection. The kit of the invention comprises one pair of specific primers, and nucleotide sequences of the specific primers are respectively as shown by SEQ ID No. 2 and 3. The kit of the invention is simple in application method, low in cost, easy inobserving a reaction result, high in sensitivity, high in specificity, capable of rapidly detecting K subgroup avian leukosis viruses, very suitable for disease monitoring, on-site emergency and clinical sample detection and suitable for large-scale popularization and application.

Description

technical field [0001] The present invention relates to the field of molecular biology, in particular to a target sequence and a fluorescent quantitative PCR primer for detecting K subgroup avian leukosis virus, and also relates to a method and a kit for detecting K subgroup avian leukosis virus using the target sequence . Background technique [0002] At present, there are no effective vaccines and drugs for the prevention, control and treatment of avian leukosis. The control of avian leukosis virus (ALV) at home and abroad is mainly to detect ALV antigens on breeders regularly, and eliminate positive chickens to achieve complete elimination. Purpose of ALVs. Subgroup K avian leukemia virus (ALV-K) is a new subtype of avian leukemia virus newly discovered in recent years. It has weak tumorigenic ability and weak replication ability in vivo and in vitro. However, avian leukosis virus is easy to mutate, and its potential threat cannot be ignored, so the detection and purifi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/701C12Q2563/107C12Q2531/113
Inventor 曹伟胜陈建严立福郑晓翠廖明
Owner SOUTH CHINA AGRI UNIV
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