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Real-time fluorescent quantitative PCR detection method and reagent kit for simian foamy virus

A real-time fluorescence quantification, foam virus technology, applied in the field of molecular biology detection of viruses, can solve problems such as the incompatibility of various detection technologies, and achieve the effects of simple and easy-to-use operating procedures, pollution prevention, and high sensitivity

Inactive Publication Date: 2010-09-15
NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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  • Abstract
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Problems solved by technology

[0010] In summary, in order to analyze the infection status of simian foamy virus in monkey groups, it is necessary to realize batch detection of monkey groups and improve the sensitivity of detection. However, many existing detection techniques are not suitable. Establishing a method suitable for batch detection of monkey groups The method of simian foam virus and the development of corresponding detection reagents have become very urgent

Method used

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  • Real-time fluorescent quantitative PCR detection method and reagent kit for simian foamy virus
  • Real-time fluorescent quantitative PCR detection method and reagent kit for simian foamy virus
  • Real-time fluorescent quantitative PCR detection method and reagent kit for simian foamy virus

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Embodiment 1

[0037] Embodiment 1, be used for the design of the primer and TaqMan probe that real-time fluorescent quantitative PCR detects to simian foamy virus (SFV)

[0038] Sequences of murine lymphocytic leukemia virus (MuLV) and human immunodeficiency virus (HIV), which are very similar to the simian foamy virus genome structure, were compared respectively, and the conserved region of the simian foamy virus pol gene (GeneBank: 6386687) was selected, and ABI PrimerExpress was used to 3.0 real-time fluorescence quantitative PCR primer design software, designed and synthesized TaqMan probes and primers. The fluorescent labeling of the probe selects FAM (5' end) as the reporter luminescent group, and NFQ (3' end) as the quenching group. The sequence is as follows:

[0039] Upstream primer (SFV-1): 5'-CAGTGTCTGGTAACTAACGCTACAA 3' (SEQ ID NO: 1 in the sequence listing);

[0040] Downstream primer (SFV 2): 5'-GGTTTTAGAGGCTTTACAGGCCTA-3' (SEQ ID NO: 2 in the sequence listing).

[0041] Ta...

Embodiment 2

[0042] Embodiment 2, the real-time fluorescent quantitative PCR detection of simian foamy virus

[0043] 1. Establishment of standard curve

[0044] 1. Construction of pGEM-T Easy-pol recombinant plasmid

[0045] 1.1 Cloning of the target gene-Nest PCR reaction

[0046] The RNA of simian foamy virus was extracted by TRIzol method, and cDNA was synthesized by reverse transcription. The reverse transcription system was as follows:

[0047] RNase-free water 7ul

[0048] Reverse transcription buffer 5ul (Promega reverse transcription system)

[0049] dNTP 4ul (each 2.5mM)

[0050] Random primer 0.5ul (Promega company reverse transcription system, the concentration is 500μg / mL)

[0051] RNA template 8ul26.17μg / mL

[0052] Reverse transcriptase 0.5ul10U / μL

[0053]

[0054] Total volume 25ul

[0055] The reaction conditions are: 37°C for 90 minutes, 72°C for 15 minutes, and 4°C for 5 minutes.

[0056] The sequence of nested primers synthesize...

Embodiment 3

[0189] Embodiment 3, the real-time fluorescent quantitative PCR detection kit of simian foamy virus

[0190] The primers and TaqMan probe mixture 360 ​​μl used for real-time fluorescent quantitative PCR detection of simian foamy virus, 5 mL of quantitative PCR reaction Mix, 500 μl of Rnase, standard (1.0×10 -7 , 1.0×10 -6 , 1.0×10 -5 , 1.0×10 -4 , 1.0×10-3 , 1.0×10 -2 , 1.0×10 -17 gradient, each gradient 25 μl), positive control 25 μl, negative control 25 μl, RNase-free water 5mL are packaged together to obtain a real-time fluorescent quantitative PCR detection kit for simian foamy virus.

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Abstract

The invention discloses a real-time fluorescent quantitative PCR detection method and a reagent kit for simian foamy virus. Primers and a TaqMan probe are designed according to the conserved regions of SFV Pol genes and are used for quantitatively detecting the number of the copied nucleic acids in the SFV of the simian lymphocytes or related biological products. Specifically, the upstream primer has the nucleic acid sequence of SEQ ID NO: 1 in the sequence table, the downstream primer has the nucleic acid sequence of SEQ ID NO: 2 in the sequence table, and the TaqMan probe has the nucleic acid sequence of SEQ ID NO: 3 in the sequence table. The real-time fluorescent quantitative PCR detection method and the reagent kit can be used for accurately detecting the infection with the SFV of the simian group and the residue of the SFV of related biological products, are very important to the control of the quality of the experimental simian group, the transmission of the SFV, the safety of the medicament use of people and the inspection and the quarantine of import and export of animals and can ensure the quality of elated biological products. The real-time fluorescent quantitative PCR detection method and the reagent kit can be used for the simian foamy virus and has broad application prospect.

Description

technical field [0001] The invention relates to a molecular biology detection method for viruses in the field of biotechnology, in particular to a real-time fluorescent quantitative PCR detection method and a kit for simian foamy virus. Background technique [0002] The source of infection of Simina Foamy Viruses (SFV) is mainly gorillas, African baboons, and vervet monkeys, which mainly exist in Africa, America, and Asia. ) Transmission among populations occurs from time to time. SFV is transmitted through bodily fluids and can be passed on to offspring. Among animal workers, SFV infection is more common than simian immunodeficiency virus (SIV) infection and has a longer incubation period. Although SFV is not pathogenic to primates, the transmission between species will cause pathogenic changes, and the infection of SFV is of great significance to humans. The research on the pathogenicity of SFV has always been concerned by scholars from various countries. Some scholars ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68G01N21/64C12N15/11
Inventor 贺争鸣栗景蕊付瑞李晓波
Owner NAT INST FOR THE CONTROL OF PHARMA & BIOLOGICAL PROD
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