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Method for detecting integrated copy number of porcine endogenous retrovirus (PERV) through fluorescence quantitative polymerase chain reaction (PCR) and application thereof

A technology of retrovirus and fluorescent quantification, which is applied to the determination/testing of microorganisms, fluorescence/phosphorescence, biochemical equipment and methods, etc., which can solve the problems of high cost and poor reaction specificity, and achieve low cost and strong specificity , good repeatability

Inactive Publication Date: 2010-09-29
FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY +1
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  • Application Information

AI Technical Summary

Problems solved by technology

Taqman probes have good specificity, but in addition to synthesizing sequence-specific primers, fluorescent probes need to be synthesized, which is costly; SYBRGreen I method is economical and only needs to synthesize sequence-specific primers, but the reaction specificity is relatively poor

Method used

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  • Method for detecting integrated copy number of porcine endogenous retrovirus (PERV) through fluorescence quantitative polymerase chain reaction (PCR) and application thereof
  • Method for detecting integrated copy number of porcine endogenous retrovirus (PERV) through fluorescence quantitative polymerase chain reaction (PCR) and application thereof
  • Method for detecting integrated copy number of porcine endogenous retrovirus (PERV) through fluorescence quantitative polymerase chain reaction (PCR) and application thereof

Examples

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Embodiment 1

[0029] Example 1, Fluorescent quantitative PCR detection of integrated copy number of porcine endogenous retrovirus

[0030] 1. Acquisition of PBMC genomic DNA

[0031] EDTA-K derived from miniature pigs 2 Anticoagulated blood (for example, can be collected from the anterior vena cava in the experiment), and peripheral blood mononuclear cells (PBMC) were separated by Ficoll-Hypaque density gradient centrifugation. The method is as follows: take 2 mL of anticoagulated blood, mix it with RPMI1640 culture medium (purchased from Sciencell, USA) 1:1, carefully add it to the liquid surface of 4 mL of human lymphocyte separation medium, centrifuge at 2000 r / min for 15 min, and use Collect the cells on the interface with a capillary pipette, put them into a centrifuge tube containing 5 mL of RPMI1640 culture solution, mix well, and centrifuge at 1500 r / min for 10 min. Carefully discard the supernatant, add 4mL RPMI1640 culture medium, blow and mix, centrifuge at 1500r / min for 10min,...

Embodiment 2

[0046] Embodiment 2, the preparation of the real-time fluorescent quantitative PCR detection kit of porcine endogenous retrovirus integration copy number

[0047] 9 μl of RealMasterMix and 1 pmol each of the upstream and downstream primers are packaged together to obtain a real-time fluorescent quantitative PCR detection kit for the integrated copy number of porcine endogenous retrovirus. When using, the user can add templates according to the needs of the experiment.

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Abstract

The invention discloses a method for detecting the integrated copy number of a porcine endogenous retrovirus (PERV) through a fluorescence quantitative polymerase chain reaction (PCR) and application thereof. In the method, the copy number of the porcine endogenous retrovirus which is integrated in a genome is detected by using SYBR Green I dye-based fluorescence quantitative PCR technology, and a PERV pol gene conservative region is taken as a detection object. The detection method has the advantages of high sensitivity, high specificity, good repeatability, rapidness, simpleness and convenience, low cost, capability of rapidly and correctly detecting the copy number of the PERV and estimating the copy number of the PERV which is integrated in a single cell genome, suitability for rapidly screening the copy number of the PERV integrated in a heteroplastic small pig donor and a porcine biological material genome and performing long-term monitoring and forward evaluation on the spreading of heteroplastic PERV, deep practical meaning and wide application prospect.

Description

technical field [0001] The invention belongs to the technical field of nucleic acid detection, and in particular relates to a fluorescent quantitative PCR detection method for the integrated copy number of porcine endogenous retrovirus and an application thereof. Background technique [0002] With the advancement of transplantation medicine and immunology research, and due to the shortage of human donor organs, pigs were selected as the most suitable xenotransplantation donors. Breeding SPF pigs can eliminate many pathogens, but not endogenous viruses. In 1997, Pateince et al. ("Infection of human cells by an endogenous retrovirus of pigs." Nat Med, 1997, 3 (3): 282-286.) first discovered porcine endogenous retrovirus (porcineendogenous retrovirus, PERV) in vitro A variety of human cells can be infected, a finding that has raised widespread concerns about the safety of xenotransplantation. PERV refers to a retrovirus that integrates into the pig cell genome in the form of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/70C12Q1/68G01N21/64
Inventor 章金刚马玉媛吴健敏冯书堂吕茂民杨勇贤
Owner FIELD OPERATION BLOOD TRANSFUSION INST OF PLA SCI ACAD OF MILITARY
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