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Digital PCR-based primers, probes and kit for blood source screening

A kit and digital technology, applied in the field of digital PCR, can solve the problems of dependence on amplification efficiency and low inhibitor tolerance, and achieve the effect of shortening the window period and improving the sensitivity

Pending Publication Date: 2020-03-27
湖南圣洲生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The most widely used and most common NAT technology is fluorescence-PCR (TaqMan technology). However, fluorescent PCR quantification requires standards and is a relative quantitative method. The result judgment depends on the amplification efficiency and the inhibitor tolerance is low.

Method used

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  • Digital PCR-based primers, probes and kit for blood source screening
  • Digital PCR-based primers, probes and kit for blood source screening
  • Digital PCR-based primers, probes and kit for blood source screening

Examples

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Embodiment 1

[0020] This embodiment is a specific application example based on the digital PCR blood source screening kit. The kit provided in this example designs amplification primers and taqman probes based on the gene sequences of HBV, HCV, and HIV to specifically detect target genes. The main criteria for screening detection sites are sequence conservation and no repetitive sequences. The HBV detection site probe is labeled FAM, the HCV probe is labeled HEX, the HIV probe is labeled ROX, and the internal standard probe is labeled CY5.

[0021] In the kit of this embodiment, the primers and probes included and their design principles are as follows:

[0022] Primer and probe design principles: use primer express 3.0.1 to design primers and probes for the screened sequence. The primer annealing temperature is about 59°C, and the probe annealing temperature is about 68°C. Dimers and hairpin structures are not easy to form. , GC content is normal, the detection amplification range is 65-250bp...

Embodiment 2

[0056] This embodiment is another specific application example of a kit for blood source screening based on digital PCR. The kit provided in this embodiment designs amplification primers and taqman probes based on the gene sequences of HBV, HCV, HIV, and TP to specifically detect target genes. The main criteria for screening detection sites are sequence conservation and no repetitive sequences. The HBV detection site probe is labeled FAM, the HCV probe is labeled HEX, the HIV probe is labeled ROX, the internal standard probe is labeled CY5, and the TP probe is labeled AlexaFluor 700.

[0057] In the kit of this embodiment, the primers and probes included and their design principles are as follows:

[0058] Primer and probe design principles: use primer express 3.0.1 to design primers and probes for the screened sequence. The primer annealing temperature is about 59°C, and the probe annealing temperature is about 68°C. Dimers and hairpin structures are not easy to form. , GC conten...

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Abstract

The invention provides a digital PCR-based primer combination, probe combination and kit including the above primer combination and probe combination for blood source screening, and a detection methodfor blood source screening by using the kit. According to the kit provided by the invention, the amplification primers and the taqman probes are designed based on the gene sequences of HBV, HCV, HIVand TP to specifically detect target genes, and the main criteria for screening detection loci are sequence conservation and no repeated sequences. The kit provided by the invention can realize absolute quantification without the need of a standard curve, the sensitivity to the detection of the HBV, the HCV, the HIV and the TP is improved by 10 times or more compared with that of fluorescence PCR,and the window phase of pathogen detection is further shortened.

Description

Technical field [0001] This application relates to the field of digital PCR technology, and in particular to a primer, probe and kit for blood source screening based on digital PCR. Background technique [0002] In order to ensure the safety of blood for clinical use and the quality of blood products, prevent cross-infection between blood supply and receiver and the health of blood collection and blood product workers, we must prevent hepatitis B virus, hepatitis C virus, human immunodeficiency virus and other diseases Infected patients enter the blood supply team to prevent the direct infusion of the pathogen-positive plasma of the aforementioned diseases into patients or the production of blood products. [0003] At present, the methods for clinical screening of various blood-borne diseases are mainly immunological diagnosis methods. However, due to the sensitivity of immunological diagnostic methods and inherent limitations, the missed detection of positive virus blood samples ...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/6851C12N15/11C12R1/93
CPCC12Q1/707C12Q1/706C12Q1/703C12Q1/6851C12Q2600/166C12Q2531/113C12Q2561/101C12Q2545/101Y02A50/30
Inventor 梁军景奉香吴东平颜进取
Owner 湖南圣洲生物科技有限公司
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