Triple real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of three simian retroviruses

A real-time fluorescent quantitative, retroviral technology, applied in microorganism-based methods, microbial assay/test, virus/phage, etc., to achieve the effect of improving specificity and improving sensitivity

Inactive Publication Date: 2016-06-01
海南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

But so far, there is no report of a kit for simultaneous detection of SIV, SRV1 and STLV1

Method used

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  • Triple real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of three simian retroviruses
  • Triple real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of three simian retroviruses
  • Triple real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of three simian retroviruses

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Experimental program
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Effect test

Embodiment 1

[0065] Synthesis of the target gene

[0066] According to the sequences of the three viruses published by GenBank, combined with bioinformatics methods, the gag gene of SIV, the 5'LTR gene of SRV1, and the conserved region of the gag gene of STLV1 were determined as the detection target sequences, and their DNA fragments were synthesized. Gene synthesis Completed by Shanghai Sangon Bioengineering Technology Services. The viral gene sequence is shown in Table 1.

[0067] Table 1: DNA sequences of the three viruses

[0068]

Embodiment 2

[0070] Synthesis of specific primers

[0071] According to the gene sequences of the three viruses synthesized, specific primers were synthesized, and the sequences are shown in Table 2.

[0072] Table 2: Specific Primer Sequences

[0073]

Embodiment 3

[0075] Preparation of positive plasmid

[0076] 1. PCR amplification of the target gene

[0077] (1) Amplify the SIV gene:

[0078] PCR reaction system: 2 μL of each primer, 15 μL of 2×TaqMasterMix, 5 μL of template, and make up to 30 μL with ultrapure water.

[0079] The reaction conditions of PCR were: reverse transcription at 50°C for 30min; hot start at 95°C for 5min, followed by 40 cycles of extension at 95°C for 15s, 55°C for 25s, and 72°C for 15s; and extension at 72°C for 10min.

[0080] (2) Amplification of SRV1 and STLV genes

[0081] The PCR system and reaction conditions are as described in (1).

[0082] 2. Preparation of positive plasmid

[0083] The 95-139bp PCR amplification products containing SIVgag gene, SRV15'LTR gene and STLV1gag gene were respectively cloned into pMD18T vector (Takara). The specific operation process is as follows:

[0084] (1) Prepare the following DNA solutions in a microcentrifuge tube, the total volume is 5 μL.

[0085] pMD18-TVe...

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Abstract

The invention discloses a triple real-time fluorescent quantitative PCR (polymerase chain reaction) detection method of three simian retroviruses. The method comprises: using conserved sequences from gene gag of simian immunodeficiency virus SIV, gene 5'LTR of Simian T-lymphotropic virus type I to synthesize their DNA fragments, designing and synthesizing three primer pairs and three probes, respectively: SIV: upper primer SI-F, lower primer SI-R and probe SI-P, SRV1: upper primer SR-F, lower primer SR-R and probe SR-P, and STLV1: upper primer ST-F, lower primer ST-R and probe ST-P, and establishing the triple real-time fluorescent quantitative PCR detection method to precisely and quantatively detect three simian retroviruses. The invention also relates to a kit for detection, having the advantages of high detection specificity, high accuracy and good sensitivity.

Description

technical field [0001] The invention belongs to the technical field of biological detection, and in particular relates to a triple real-time fluorescent quantitative PCR detection method for three monkey retroviruses. Background technique [0002] Simian immunodeficiency virus (SIV), also known as African green monkey virus (African Green Monkey virus), is a retrovirus that affects at least 33 species of African primates. SIV has high homology with HIV virus in gene sequence. After non-human primates are infected with SIV, they will have the pathogenesis and clinical symptoms similar to AIDS, and the immune system damage and multiple systems may appear after infection with the virus. Lesions of multiple organs and tissues, SIV is widely distributed in rhesus monkeys, and can affect multiple systems including the immune system. [0003] Simian type D retrovirus (simian type-Dretrovirus, SRV) belongs to Retroviridae, D-type retrovirus genus, and simian immunodeficiency virus ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68C12N15/11C12N15/70C12R1/93
CPCC12N15/70C12N2800/101C12Q1/6851C12Q1/702C12Q2600/16C12Q2531/113C12Q2545/113C12Q2563/107C12Q2537/143
Inventor 李丹丹高慎阳徐义刚邱索平
Owner 海南出入境检验检疫局检验检疫技术中心
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