Specific primer, probe and kit for real-time fluorescence detection of xenotransplantation donor pig multiple viruses

A xenotransplantation and real-time fluorescence technology, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., can solve problems such as strong infectivity, improve specificity, reduce missed detection, and improve postoperative Survival effect

Pending Publication Date: 2022-06-28
杭州时轮医药科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But the recombinant form of PERV-A / C is highly infectious in human cell culture

Method used

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  • Specific primer, probe and kit for real-time fluorescence detection of xenotransplantation donor pig multiple viruses
  • Specific primer, probe and kit for real-time fluorescence detection of xenotransplantation donor pig multiple viruses
  • Specific primer, probe and kit for real-time fluorescence detection of xenotransplantation donor pig multiple viruses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0063] Example 2 Specificity

[0064] The present invention aims at designing specific primers and probes, which can detect PERV, SI and PHEV respectively without cross-reaction, which shows that the kit has good specificity.

Embodiment 3

[0065] The establishment and optimization of embodiment 3 reaction system

[0066] The reaction conditions were determined by repeated experiments: 10 min at 50°C for 1 cycle; 10 min at 94°C for 1 cycle; and 45 cycles at 94°C for 15s and 55°C for 45s.

Embodiment 4

[0067] Embodiment 4 detection method steps

[0068] According to relevant domestic and foreign literature reports, the detectable specimens include: serum (applicable to PERV, HEV), peripheral whole blood (PERV), respiratory swab samples and lung tissue samples (applicable to SI), etc. Specimens can be used for testing immediately, or can be stored at -70°C for testing with a storage period of 6 months.

[0069] (l) RNA extraction: Use MagNA Pure LC and Roche MagNA Nucleic Acid Extraction and Purification Kit (Roche CatN; 05323738001), or nucleic acid extraction kits produced by other companies, or personal nucleic acid extraction and purification methods, in the recommended operating procedures Next, high-purity RNA can be extracted. The RNA internal extraction reference (IEC: BIOLINE BIO38041) was added to the sample before extraction as a quality control.

[0070] (2) RT-PCR reaction:

[0071] Make the reaction mixture according to the following formula:

[0072] ...

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Abstract

The invention discloses a specific primer, a probe and a kit for real-time fluorescence detection of multiple viruses of a xenotransplantation donor pig, which are characterized in that nucleic acid sequences of a porcine endogenous retrovirus (PERV), a swine influenza virus (SI) and a swine hepatitis E virus (PHEV) are compared and analyzed, and the nucleic acid sequences of the PERV-GAG gene 1332-1430 segment (three types of PERV-ABC are highly conservative) and the SI-HA gene are respectively compared and analyzed to obtain the real-time fluorescence detection of multiple viruses of the xenotransplantation donor pig. The method comprises the following steps of: selecting a high-conservative section without a secondary structure by taking a PHEV-ORF3 gene as an amplification target site, and determining a plurality of pairs of primers and probes suitable for real-time fluorescent quantitative Real Time-PCR (RT-PCR or qPCR) by utilizing primer analysis software and manual judgment according to the basic principle of primer probe design and through multiple times of experimental optimization. The invention further discloses a corresponding kit and method for rapid screening and quantitative detection. The present invention provides methods for simultaneous screening of donor porcine endogenous retrovirus (PERV), swine influenza virus (SI), and swine hepatitis E virus (PHEV).

Description

technical field [0001] The invention belongs to the field of nucleic acid detection, and provides a real-time fluorescent detection method for xenotransplantation donor pig multiple viruses, which can simultaneously screen donor pig endogenous retrovirus (PERV), swine influenza virus (SI ) and methods for porcine hepatitis E virus (PHEV) RNA. [0002] technical background [0003] At present, the biggest problem facing organ transplantation is the shortage of donors. Pig-derived xenotransplantation provides hope for most patients who die due to organ failure and lack of donor organs. However, with the transplantation of pig donor organs into humans, some pig-derived viruses may also be transmitted to humans and cause disease. Porcine cytomegalovirus (PCMV) and porcine lymphatic herpesvirus (PLHV) are largely species-specific. Sexual, usually does not infect human cells. Porcine ADV and PCV2 generally do not cause severe disease and have not been reported to spread to humans...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/11
CPCC12Q1/702C12Q1/701C12Q1/707C12Q1/686C12Q2600/16C12Q2561/113C12Q2563/107C12Q2545/114
Inventor 李旦史金阳郑炜万建峰
Owner 杭州时轮医药科技有限公司
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