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Application of SNP (Single Nucleotide Polymorphism) marker and SNP loci as well as primers, probes and detection kit of SNP marker and SNP loci

A site and marker technology, used in the determination/inspection of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., can solve the problems of low accuracy and sensitivity, and influence on the accuracy of results, and achieve low-cost urine collection. , good monitoring effect, high detection sensitivity

Pending Publication Date: 2021-12-03
成都仕康美生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] 3) If the puncture needle does not collect the lesion tissue part of the graft, it will have a great impact on the accuracy of the results, so the accuracy and sensitivity are not high

Method used

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  • Application of SNP (Single Nucleotide Polymorphism) marker and SNP loci as well as primers, probes and detection kit of SNP marker and SNP loci
  • Application of SNP (Single Nucleotide Polymorphism) marker and SNP loci as well as primers, probes and detection kit of SNP marker and SNP loci
  • Application of SNP (Single Nucleotide Polymorphism) marker and SNP loci as well as primers, probes and detection kit of SNP marker and SNP loci

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0127] A SNP marker for assessing the risk of graft injury and rejection after liver transplantation, the SNP markers are SNP sites rs2293840, rs2293841, rs2293843, rs163792, rs1569959, rs2250911, rs1903858, rs2293871, rs2293877, rs817363, rs22938990, 、rs704326、 rs2249102、rs2294047、rs2295499、rs2294111、rs208244、rs572496、rs2294822、 rs2295167、rs2295166、rs2295164、rs1321666、rs2295632、rs2295147、rs2974、 rs1547093、rs2295072、rs1322775、rs2255090、rs2294992、rs1022811、rs786906、 rs1738051、rs2294897、rs2294895 A combination of , rs2294886, rs2294871 and rs926587.

[0128] A kit for assessing the risk of graft injury and rejection after liver transplantation, comprising primers whose sequences are shown in SEQ ID No.1-80 in Table 1 and probes whose sequences are shown in SEQ ID Nos.81-160 Needle, ddPCR TM ProbeSupermix and ultrapure water;

[0129] The 5' ends of the probes are respectively connected with FAM fluorescent groups, and the 3' ends of the probes are labeled with NFQ and MGB.

...

Embodiment 2

[0143] Using the kit provided by the invention to assess the risk of graft injury and rejection after liver transplantation

[0144] 1. Extraction of total free DNA in urine

[0145] 10ml of urine was collected from a recipient 8 weeks after liver transplantation, and the urine was centrifuged at 3000rpm for 5min.

[0146] Take 4ml of centrifuged urine supernatant, use QIAamp Circulating NucleicAcid Kit from QIAGEN to extract total free DNA, adjust the DNA concentration to 10-30ng / ul.

[0147] 2. Prepare digital PCR reaction droplets and perform digital PCR reaction

[0148] 2.1. Configure the digital PCR reaction system, prepare digital PCR reaction droplets, and perform PCR reaction; the detection system of 40 SNP sites, a total of 40 tubes, the reaction preparation is as follows:

[0149] Table 2

[0150]

[0151] Among them, the primers used to detect SNP sites include upstream and downstream primers, each 0.125 μL, a total of 0.25 μL; two probes, each 0.125 μL, a to...

Embodiment 3

[0170] Include 100 cases of liver transplant recipients diagnosed with transplant rejection and 100 cases of liver transplant recipients diagnosed with normal grafts, collect their urine according to Example 2, extract the total free DNA in the urine, and prepare digital PCR reaction microbiota. After the reaction, analyze each SNP site, calculate the GcfDNA supply-to-receiver ratio, and set 2.5% as the reference value to distinguish transplant rejection (GcfDNA supply-to-receiver ratio>2.5%) and normal graft group (GcfDNA supply to recipient ratio ≤ 2.5%) patients.

[0171] Use MedCalc software to analyze, obtain the ROC curve ( figure 1 ), AUC=0.88, sensitivity=82.86%, specificity=72.41%.

[0172] The kit of the invention can timely, accurately and specifically reflect the health status of the liver graft, and evaluate the graft injury and rejection risk after liver transplantation.

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Abstract

The invention relates to the technical field of biology, discloses an application of an SNP (Single Nucleotide Polymorphism) marker and SNP loci as well as primers, probes and a detection kit of the SNP marker and the SNP loci, and particularly provides an application of 40 SNP loci in evaluating transplanted plant injury and rejection risks after liver transplantation. By detecting the 40 specific SNP loci of total free DNA of a transplanted receptor, a GcfDNA donor-receptor ratio is obtained; and the degree of transplantation rejection is rapidly and accurately judged according to the GcfDNA donor-receptor ratio, no invasive detection is needed, accuracy and sensitivity are high, and a simple and effective auxiliary means is provided for clinical detection of transplantation injury and rejection risks after organ transplantation.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of SNP markers, SNP sites, primers, probes and detection kits. Background technique [0002] At present, the health monitoring of grafts after solid organ transplantation often uses blood drawing for liver function tests, or tissue collection by puncture needles for pathological examination. [0003] For routine blood drawing function tests, the sensitivity and specificity of various indicators such as creatinine, ALT, AST, bilirubin, etc. are not high, and cannot accurately reflect the health status of the graft. [0004] For the current gold standard tissue biopsy, although it can directly reflect the health status of the graft. But there are the following major disadvantages: [0005] 1) Invasive detection, which causes great pain to the patient and damages the graft at the same time; [0006] 2) When the abnormality is detected, the substantial damage of the gr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2531/113C12Q2563/107C12Q2563/159
Inventor 魏亮
Owner 成都仕康美生物科技有限公司
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