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RT-LAMP detection primer set and kit for subgroup K avian leukosis virus

A technology of avian leukosis virus and RT-LAMP, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of research lag and achieve the effects of short time consumption, good specificity and low cost

Inactive Publication Date: 2017-06-20
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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  • RT-LAMP detection primer set and kit for subgroup K avian leukosis virus
  • RT-LAMP detection primer set and kit for subgroup K avian leukosis virus
  • RT-LAMP detection primer set and kit for subgroup K avian leukosis virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Establishment of Subgroup Avian Leukosis Virus RT-LAMP Detection Method

[0038] 1 Experimental materials

[0039] strains, cells

[0040] 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.

[0041] 2 Experimental methods and results

[0042] ...

Embodiment 2

[0055] The specificity of the RT-LAMP detection method of embodiment 2ALV-K

[0056] Using the optimized reaction system, ALV-K, ALV-A, ALV-B, ALV-J, ALV-E were used as templates to confirm the intraspecies specificity of RT-LAMP assay. Then ALV-K, NDV, REV, AIV, MDV, IBV, and DF-1 were used as templates to confirm the interspecies specificity of RT-LAMP assay. Such as Figure 5 with Image 6 As shown, the real-time fluorescence method results show that RT-LAMP primers can only specifically amplify ALV-K, and other templates are negative; the colorimetric method results show that only ALV-K is positive in green, and the rest are negative in orange , which proves that the method has strong specificity.

Embodiment 3

[0057] The sensitivity of the RT-LAMP detection method of embodiment 3ALV-K

[0058] The positive recombinant plasmid was diluted 10 times, from 8.4×10 10 copies / μL to 8.4×10 2 copies / μL, RT-LAMP reaction was performed with optimized reaction conditions. Design a pair of primers (F: 5'-ACGCTTTCAGATTGGTCC-3' (SEQ ID NO.7); R: 5'-AAGCGAGAACCTGTCTGTGC-3' (SEQ ID NO.8)), target gene 182bp, with the same diluted positive recombinant plasmid As a template, a conventional PCR reaction was performed to compare the sensitivity of the two detection methods. The result is as Figure 7 shown. RT-LAMP can detect at least 8.4×10 4 copies / μL. according to Figure 8 As a result, ordinary PCR can detect at least 8.4×10 3 copies / μL.

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Abstract

The invention discloses an RT-LAMP primer set and kit for a subgroup K avian leukemia virus. The RT-LAMP detection primer set for rapidly detecting the subgroup K avian leukemia virus comprises a pair of external primers and a pair of internal primers, and is designed according to a gp85 region of a subgroup K avian leukemia virus sequence. The RT-LAMP detection primer group for the subgroup K avian leukemia virus obtained by screening can specifically amplify ALV-K, and the intraspecies and interspecies specificity is very good; at least 8.4 * 10<4> copies / mu L of nucleic acid can be detected in a reaction, and the RT-LAMP detection method of ALV-K is simple in operation, low in cost, and short in time-consumption, and is suitable for rapid clinical detection of ALV-K.

Description

technical field [0001] The invention belongs to the technical field of molecular detection, and in particular relates to an RT-LAMP detection primer set and kit for subgroup K 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 is the most rout...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12N15/11
CPCC12Q1/702C12Q1/6844C12Q2531/119C12Q2521/107C12Q2563/107
Inventor 曹伟胜杨素赵海燕陈轩黄海超赵福振邵建宏沙才华
Owner 珠海出入境检验检疫局检验检疫技术中心
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