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New purpose of AMPH for diagnosing hepatic failure, reagent kit and application of reagent kit for diagnosing hepatic failure

A liver failure and kit technology, applied in the fields of biomedicine, medical testing, and clinical medicine, can solve problems such as liver metabolism, detoxification function reduction, systemic cytokine storm, liver cell necrosis, etc. small effect

Inactive Publication Date: 2019-09-13
杭州笙源生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Significant abnormalities of these indicators usually occur in patients with obvious liver cell necrosis, systemic cytokine storm, liver metabolism and detoxification function are significantly reduced, and the disease enters a stage of rapid progression, which is difficult to control and causes great difficulties in treatment.

Method used

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  • New purpose of AMPH for diagnosing hepatic failure, reagent kit and application of reagent kit for diagnosing hepatic failure
  • New purpose of AMPH for diagnosing hepatic failure, reagent kit and application of reagent kit for diagnosing hepatic failure
  • New purpose of AMPH for diagnosing hepatic failure, reagent kit and application of reagent kit for diagnosing hepatic failure

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] experimental method

[0045] 1. Peripheral blood mononuclear cell isolation

[0046] 1. Draw 8mL of whole blood from the patient, let it stand for about 30 minutes, and pre-cool the centrifuge to 4°C.

[0047]2. Using a density gradient centrifuge, centrifuge at 4°C and 3000 rpm for 5 minutes. The blood cell pellet was fully resuspended with 5mL sterile 0.1M PBS buffer, mixed evenly to form a blood cell suspension, and then slowly added to the centrifuge tube of Ficoll-paqueTM PLUS medium lymphocyte separation medium to form layers naturally. Set the temperature of the centrifuge to 20°C, the speed to 2500 rpm, speed up to 9, speed down to 1, and centrifuge for 20 minutes.

[0048] 3. Aspirate the middle buffy coat layer, add 10 mL of PBS buffer, centrifuge at 1200 rpm at 4°C for 4 minutes, discard the supernatant. This step was repeated 3 times.

[0049] 4. Blot the residual PBS buffer with a pipette gun, and add 1 mL of cell lysis solution TRIzol to completely lys...

Embodiment 2

[0065] Real-time fluorescent quantitative PCR (qRT-PCR) was used to detect the expression of AMPH mRNA in liver failure, high-risk groups of liver failure, liver cirrhosis, chronic hepatitis B and healthy controls.

[0066] Experimental procedure

[0067] qRT-PCR uses a two-step method for experimental operation. Separation of peripheral blood mononuclear cells and extraction of total RNA were carried out in the same manner as in Example 1, and the next step of the experimental operation was carried out after the mRNA concentration was tested.

[0068] 1. Reverse transcription: QuantiTect Reverse Transcription kit (Qiagen, CA, USA) was used for reverse transcription, and cDNA was obtained by following the instructions of the kit.

[0069] 2. Take the above cDNA template for PCR amplification: the PCR reagent uses TB Green reagent from Takara Company, and the operation is according to the kit instructions.

[0070] figure 2 Peripheral blood mononuclear samples from liver fa...

Embodiment 3

[0071] Example 3 The kit includes: AMPH amplification primers such as the primer pair shown in the front primer of SEQ ID NO.1 and the rear primer of SEQ ID NO.2, and the internal reference such as the front primer of SEQ ID.NO.3 and the rear primer of SEQ ID NO.4 The primer pair shown: 2.5U / μl PolyA polymerase, reverse transcriptase, 5× reverse transcription buffer, RT mixture, 5ml RNase-free water, qPCR mixture, adsorption column, cell lysate. Application of AMPH mRNA kit in the diagnosis of pigs with liver failure

[0072] Animal model: 30 male Chinese young pigs (8-10 kg) were randomly divided into two groups, 15 in each group.

[0073] Experimental group: Each young pig was injected with D-gal 1.5g / kg into the jugular vein to create a liver failure model.

[0074] Control group: inject the same amount of normal saline without AMPH.

[0075] The peripheral blood mononuclear cells and serum samples of the experimental group and the control group were separated, and the to...

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Abstract

The invention discloses a new purpose of AMPH for diagnosing hepatic failure, a reagent kit and an application of the reagent kit for diagnosing the hepatic failure. A gene AMPH is screened through combination of high-flux mRNA sequencing with a bioinformatics method; and further through a fluorescence quantitative PCR method, the situation that AMPH molecules have good relevance to the hepatic failure is verified, so that the AMPH can be used for diagnosing the hepatic failure, and has important clinical application value. The invention further discloses a reagent kit for diagnosing the hepatic failure. The reagent kit comprises a detection reagent containing the AMPH. Through uniting of high-flux sequencing and qPCR verification, the inventor finds that the AMPH presents characteristic high expression in various samples of patients suffering from the hepatic failure, has good relevance to diseases, is stable and reliable, can timely reflect the disease state of the patients sufferingfrom the hepatic failure, can early diagnose the hepatic failure, can direct a doctor to perform early treatment and can reduce case fatality rate.

Description

technical field [0001] The invention relates to the technical fields of clinical medicine, biomedicine and medical testing, in particular to a new application of detecting AMPH molecules for diagnosing liver failure, a kit and an application of the kit for diagnosing liver failure. Background technique [0002] Liver failure is a kind of disease caused by extensive necrosis of the liver caused by various reasons. Except for orthotopic liver transplantation, there is currently no specific treatment. However, due to the severe shortage of donor livers, a large number of patients died while waiting for liver transplantation. The current clinical method for diagnosing liver failure is based on the traditional coagulation indicators of total bilirubin in biochemical indicators, such as the international normalized ratio of coagulation, prothrombin activity and so on. Significant abnormalities of these indicators usually occur in patients with obvious liver cell necrosis, system...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/158C12Q2600/166
Inventor 李君陈新李江辛娇娇
Owner 杭州笙源生物科技有限公司
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