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K sub-group ALV (avian leukosis virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection primer group and kit

An avian leukemia virus and fluorescence quantitative technology, which is applied in the determination/inspection of microorganisms, microorganisms, and recombinant DNA technology, etc., can solve problems such as research lag, and achieve the effects of high sensitivity, good repeatability and strong specificity.

Inactive Publication Date: 2017-05-31
珠海出入境检验检疫局检验检疫技术中心
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The classic exogenous avian leukosis virus subgroups ALV-A, ALV-B, and ALV-J are relatively mature in detection and purification, while the ALV-K subgroup, which was only discovered and named in 2008, is relatively mature in related aspects. Research is very lagging

Method used

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  • K sub-group ALV (avian leukosis virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection primer group and kit
  • K sub-group ALV (avian leukosis virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection primer group and kit
  • K sub-group ALV (avian leukosis virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Establishment of Fluorescent Quantitative RT-PCR Detection Method for K Subgroup Avian Leukosis Virus

[0031] 1 Experimental materials

[0032] strains, cells

[0033] American ATCC chicken embryo fibroblast cell line (DF-1), A subgroup avian leukemia RAV-1 strain (GenBank accession number: M19113), B subgroup avian leukemia RAV-2 strain (GenBank accession number: M14902), J subgroup Group avian leukemia strain HPRS-103 (GenBank accession number: Z46390), E subgroup avian leukemia strain RAV-0 (GenBank accession number: M12172), K subgroup avian leukemia strain GDFX0601 (GenBank accession number: KP686142.1), poultry Reticuloendotheliosis virus (REV), chicken Marek's disease virus (MDV), avian influenza virus (AIV), chicken infectious bronchitis virus (IBV) are the key experiments for the creation of veterinary vaccines by the Ministry of Agriculture of the Veterinary School of South China Agricultural University Room preservation.

[0034] 2 Experimental ...

Embodiment 2

[0054] Embodiment 2 specificity experiment

[0055] Using ALV-K, ALV-A, ALV-B, ALV-J, and ALV-E as templates, quantitative RT-PCR was carried out to detect its intraspecies specificity. Then ALV-K, NDV, REV, AIV, MDV, IBV, and DF-1 were used as templates to detect their interspecies specificity. The results showed that only ALV-K showed an "S"-type amplification curve, and other subgroups of ALV and other viruses did not show amplification curves, which proved that the method was highly specific and could effectively detect ALV-K, and Can be distinguished from other viruses (see image 3 and Figure 4 ).

Embodiment 3

[0056] Embodiment 3 Sensitivity experiment

[0057] positive recombinant plasmid from 8.4 × 10 10 Copies / μL starts with a 10-fold serial dilution, and uses each dilution as a template to perform a fluorescent quantitative RT-PCR reaction. Design a pair of primers (F: 5'-ACGCTTTCAGATTGGTCC-3' (SEQ ID NO.6); R: 5'-AAGCGAGAACCTGTCTGTGC-3' (SEQ ID NO.7)), target gene 182bp, positive recombination with the same dilution The plasmid was used as a template, and the conventional PCR reaction was carried out to compare the sensitivity of the two detection methods. The results showed that fluorescence quantitative RT-PCR can detect at least 8.4×10 1 Copies / μL of nucleic acid, ordinary PCR can only detect a minimum of 8.4×10 3 copies / μL, it proves that the sensitivity of TaqMan probe fluorescent quantitative RT-PCR is 100 times that of ordinary PCR, and this method is suitable for clinical detection of ALV-K and chicken farm purification ( Figure 5 and Figure 6 ).

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Abstract

The invention discloses a K sub-group ALV (avian leukosis virus) fluorescent quantitation RT-PCR (reverse transcription-polymerase chain reaction) detection primer group and kit. The used primer pair and a probe are designed according to the gp85 segment of the K sub-group ALV sequence; the ALV-K can be specifically amplified; the intraspecific and interspecific specificity is very good; the 8.4*10 copies / muL viruses can be detected to the lowest degree; the sensitivity is 100 times of that of the ordinary PCR; the intra-assay variation coefficient and the inter-assay variation coefficient are lower than 5 percent. The result proves that the specificity is high; the sensitivity is high; the repeated stability is high; the amplification efficiency is high; the kit can be used for clinic ALV-K detection.

Description

technical field [0001] The invention belongs to the field of molecular detection, and in particular relates to a fluorescent quantitative RT-PCR detection primer set and a kit for K subgroup avian leukemia virus. Background technique [0002] Avian leukemia viruses (ALVs) are members of Retroviridae and Oncoviridae. All benign and malignant tumors caused by this virus group are collectively called avian leukemia (AL). (Yin Zhen, 1997). Avian leukemia is listed as one of the national priority animal diseases in 2012-2020 (Gao Hongbin, 2012). Tumors induced by avian leukemia virus mainly include leukemia, connective tissue tumors, epithelial tumors, endothelial tumors and other related tumors. Leukemia can be further divided into lymphocytic leukemia (Lymphoid Leucosis, LL), erythroblastosis EB), myeloblastosis (Myeloblastosis, MB) and myelocytic leukemia (Myelocytomatosis, ML) (Purchase et al., 1984; Hofstad, 1980). [0003] At present, virus isolation and identification i...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12R1/93
CPCC12Q1/702C12Q1/6851C12Q2531/113C12Q2563/107C12Q2521/107
Inventor 杨素陈轩苏惠龙邵建宏黄海超赵福振沙才华赵海燕
Owner 珠海出入境检验检疫局检验检疫技术中心
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