RT-RAA fluorescence method detection primer pair, kit and detection method for J subtype avian leukosis virus gp85 gene

An avian leukemia virus, RT-RAA technology, applied in the field of molecular biology, can solve problems such as false positives, and achieve the effects of strong specificity, low false positive rate and low cost

Pending Publication Date: 2022-01-14
SOUTH CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

ELISA diagnostic method for ALV-J was used to detect group-specific antigens, but false positives occurred due to detection of endogenous virus group-specific antigens

Method used

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  • RT-RAA fluorescence method detection primer pair, kit and detection method for J subtype avian leukosis virus gp85 gene
  • RT-RAA fluorescence method detection primer pair, kit and detection method for J subtype avian leukosis virus gp85 gene
  • RT-RAA fluorescence method detection primer pair, kit and detection method for J subtype avian leukosis virus gp85 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Initial Primer Design

[0051] For the gp85 gene sequence of J subtype avian leukosis virus in the NCBI database (GenBank accession numbers: Z46390, MT175600, MN066150, MN066151, MN066152, MK940585, MN066140, JQ935966, JX848322, KC149971, JF932004, 9K9U1 are used as reference for comparison analysis) Conserved region, after homology analysis, design primer probe (Probe) as follows:

[0052] Probe: CTCTCTCCTAACTTTACCACCTGGATAACATATGGGCCGAACATTACG (shown in SEQ ID NO.5); the probe is modified as follows: CTCTCTCCTAACTTTACCCACCTGGATAACA / i6FAMdT / / THF / / iBHQ1dT / GGGCCGAACATTACG;

[0053] F-1: CCCTGTTATTCTAGGACAATTGACTATG (shown in SEQ ID NO.6);

[0054] F-2: CACCAGGTATTTTTCTTGATTTGTGGGG (shown in SEQ ID NO.7);

[0055] F-3: AGGGCCCTGTTATTCTAGGACAATTGACTATG (shown in SEQ ID NO.8);

[0056] R-1: CACACTCCATAGCTGTAGCTCATCACCGCAG (shown in SEQ ID NO.9);

[0057] R-2: CTCCATAGCTGTAGCTCATCACCGCAG (shown in SEQ ID NO.10);

[0058] R-3: CATAGCTGTAGCTCATCACCGCAGTCAGGCGAG (shown in ...

Embodiment 2

[0080] Validation of Primer Pairs for Detection of J Subtype Avian Leukosis Virus gp85 Gene RT-RAA

[0081] The primers screened in Example 1 verified the F1R1 amplified fragment, the template was the J subtype avian leukosis virus SCAU1903 nucleic acid (shown in SEQ ID NO.4), and the reaction system was: forward primer F1 (10 μM) 1.0 μL, reverse To primer R1 (10 μM) 1.0 μL, ddH 2 O 6.0 μL, 2x Es Taq MasterMix 10.0 μL, template 2 μL. The reaction program was: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 30 s, renaturation at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles; extension at 72°C for 2 min. The obtained product was analyzed by 1% agarose gel electrophoresis.

[0082] The result is as Figure 4 As shown, the screened primer pair amplifies the slice pair consistent with the expected result, the size is 148bp, and the swimming lanes are Marker, J subtype avian leukosis virus SCAU1903 nucleic acid, ddH 2 O negative control.

Embodiment 3

[0084] A kit for detecting J subtype avian leukosis virus, the kit is composed of the following components: RT-RAA reaction dry powder, forward primer F1 screened in Example 1, reverse primer R1 screened in Example 1, Example 1 1 Modified probe JP, liquid A, liquid B, positive control substance and negative control substance after screening; wherein the molar ratio of forward primer, reverse primer and probe is 1:1:1; positive control substance contains J subtype avian leukosis virus SCAU1903 nucleic acid (shown in SEQ ID No.4), negative control substance is ddH 2 O.

[0085] The composition of RT-RAA reaction dry powder is: MLV reverse transcriptase, recombinant enzyme, single-chain binding protein, polymerase, ATP, dNTP Mix, magnesium chloride (MgCl 2 ); A liquid composition is: polyethylene glycol (PEG); B liquid composition is: magnesium acetate (MgAc2); The RT-RAA reaction dry powder, A liquid and B liquid are purchased from Nanning Zhuangbo Biotechnology Co., Ltd. Comp...

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Abstract

The invention relates to the technical field of molecular biology, in particular to an RT-RAA fluorescence method detection primer pair, a kit and a detection method for a J subtype avian leukosis virus gp85 gene. The primer pair comprises a forward primer and a reverse primer; and the nucleotide sequence of the forward primer is as shown in SEQ ID NO.1, and the nucleotide sequence of the reverse primer is as shown in SEQ ID NO.2. The primer pair can specifically amplify the J subtype avian leukosis virus gp85 gene, the J subtype avian leukosis virus can be specifically detected by utilizing the primer pair, the detection period is shorter, and the specificity is higher.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a RT-RAA fluorescence detection primer pair, a kit and a detection method for the gp85 gene of J subtype avian leukosis virus. Background technique [0002] Avian leukemia is caused by the avian leukosis virus (Avianleukosisvirus, ALV) of the genus Avian Retrovirus in the Retroviridae family. Erythroblastic avian leukemia, myeloblastic avian leukemia, connective tissue tumors, nephroma, nephroblastoma, and various other epithelial, endothelial, and neurological tumors. In recent years, myeloid avian leukemia has shown a gradual upward trend, replacing lymphoid avian leukemia as the main type. [0003] The ALV of avian leukemia that is prevalent in various parts of the country is mainly A, B, and J subgroups. Among these three subgroups, J type infection is the most serious, and it is found in laying hens, broiler chickens and chickens of many local strains. found. Ho...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/6844C12N15/11C12R1/93
CPCC12Q1/702C12Q1/6844C12Q2521/107C12Q2521/507C12Q2522/101C12Q2531/119C12Q2563/107
Inventor 谢青梅张新珩巫秀红陈丽怡姚梓淇
Owner SOUTH CHINA AGRI UNIV
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