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Hepatitis B virus miR-3 and human liver specific miR-122 triple fluorescent quantitative PCR detection kit

A hepatitis B virus, fluorescence quantitative technology, applied in medical detection and biological fields, can solve the problems of inability to absolutely quantify serum microRNA molecule copy concentration, low efficiency of single-color fluorescence PCR, etc., to achieve the existence of monitoring false negatives and good specificity , the effect of improving efficiency

Active Publication Date: 2019-03-26
THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a triple fluorescent quantitative PCR detection kit for hepatitis B virus miR-3 and human liver-specific miR-122, which solves the low efficiency of single-color fluorescent PCR of microRNA detection kits in the prior art and cannot be absolutely quantitative Technical issues of serum microRNA molecule copy concentration

Method used

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  • Hepatitis B virus miR-3 and human liver specific miR-122 triple fluorescent quantitative PCR detection kit
  • Hepatitis B virus miR-3 and human liver specific miR-122 triple fluorescent quantitative PCR detection kit
  • Hepatitis B virus miR-3 and human liver specific miR-122 triple fluorescent quantitative PCR detection kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Example 1.microRNA extraction

[0064] (1) Take 100 μl of serum / plasma and add it to a 1.5ml centrifuge tube, add 0.5ml of Trizol reagent to each tube, pipette the tip repeatedly to mix until the protein is completely dissolved, add 5 μl of 5nmol / L internal reference to each tube, mix well, and place at room temperature for 10 minute.

[0065] (2) Add 100 μl of chloroform to each tube, shake vigorously for 15 seconds, and place at room temperature for 3 minutes.

[0066] (3) Centrifuge at 12000×g for 15 minutes at 4°C, pipette 0.3ml of the supernatant into a new 1.5ml centrifuge tube, add 0.3ml of isopropanol, mix well, and let stand at room temperature for 10 minutes.

[0067] (4) Put the small molecule RNA adsorption column in a 2ml collection tube, transfer all the RNA isopropanol mixture to the small molecule RNA adsorption column, and centrifuge at 8000×g for 1 minute.

[0068] (5) Discard the filtrate, put it back into the column, add 0.5ml washing solution 1 to...

Embodiment 2

[0070] Example 2. microRNA reverse transcription

[0071] (1) Take 8 μl of 2.5×reverse transcription mixture and 0.5 μl of 10 μmol / L reverse transcription primer mixture into a 0.2 ml RNase-free PCR thin-walled tube. (wherein the 10 μmol / L reverse transcription primer mixture is a mixture of miR-3, miR-122 and C.elegansmiR-39 reverse transcription primers, and the concentration of the three is 10 μmol / L)

[0072] (2) Add 11.5 μl of the extracted microRNA into the above mixture, and mix well by pipetting.

[0073] (3) Put it into a PCR machine for reverse transcription, the conditions are: 42° C. for 30 minutes, 70° C. for 5 minutes. The obtained cDNA was stored in an ice box for later use.

Embodiment 3

[0074] Example 3. Triple fluorescent quantitative PCR

[0075] (1) Take 25 μl of 2×PCR mixed solution, 5 μl of 2 μmol / L probe mixed solution, and 15 μl of deionized water into a 0.2 ml 8-section PCR tube. (The 2 μmol / L probe mixture is a mixture of miR-3, miR-122 and C.elegans miR-39 probes, and the concentration of the three is 2 μmol / L)

[0076] (2) Add 5 μl of reverse transcribed cDNA to each tube and mix well.

[0077] (3) Put the PCR reaction tube into the fluorescent quantitative PCR amplification instrument, and set the name of the sample to be tested and the concentration of the quantitative reference product in the corresponding order

[0078] (4) Selection of fluorescence detection channel: FAM channel was selected to detect miR-3 by software, HEX was selected to detect internal reference, and Cy5 channel was selected to detect miR-122.

[0079] (5) The reaction conditions of fluorescent quantitative PCR are: 95° C. for 3 minutes, enter cycle: 95° C. for 12 seconds...

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Abstract

The invention provides a hepatitis B virus miR-3 and human liver specific miR-122 triple fluorescent quantitative PCR detection kit. The hepatitis B virus miR-3 and human liver specific miR-122 triplefluorescent quantitative PCR detection kit comprises a hepatitis B virus miR-3 reverse transcription primer as shown by SEQ ID NO. 1, a fluorescent probe as shown by SEQ ID NO. 2 and PCR primers as shown by SEQ ID NO. 3 and NO. 4, and can further comprise a human liver specific miR-122 reverse transcription primer as shown by SEQ ID NO. 5, a fluorescent probe as shown by SEQ ID NO. 6 and PCR primers as shown by SEQ ID NO. 7 and NO. 8, and can further comprise an internal reference reverse transcription primer as shown by SEQ ID NO. 9, a fluorescent probe as shown by SEQ ID NO. 10, and PCR primers as shown by SEQ ID NO. 11 and NO. 12. The kit uses a triple fluorescent quantitative PCR method to detect the concentration of hepatitis B virus miR-3 and human liver specific miR-122 in trace (100 micro liters) serum or plasma. The detection method is specific, efficient and sensitive. The kit can be used for monitoring the replication situation of the heptatitis B virus, evaluating the curative effect and prognosis of an antiviral drug and evaluating the liver injury and has an important clinical significance.

Description

technical field [0001] The invention relates to the field of biological and medical detection, in particular to a kit for detecting hepatitis B virus miR-3 and human liver-specific miR-122 by triple fluorescence quantitative PCR. Background technique [0002] HBV miR-3 is a specific microRNA encoded by the HBV genome, which controls the replication of HBV. The concentration of HBV miR-3 in serum or plasma can be used to evaluate the replication of hepatitis B virus, and can also be used to evaluate the efficacy and prognosis of antiviral therapy (Xi Yang et al.Hepatitis BVirus-Encoded MicroRNA Controls Viral Replication.Journal of Virology. 2017 Volume 91 Issue 10 e01919-16). miR-122 is a microRNA specifically expressed in human liver cells, and the concentration of miR-122 in serum can be used to evaluate the damage degree of human liver cells caused by virus activity. Both of these two microRNAs can be used as a new biological indicator for clinical detection and have im...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/686C12N15/113
CPCC12Q1/686C12Q1/706C12Q2600/178C12Q2563/107C12Q2545/114C12Q2537/143
Inventor 张旻龚国忠肖新强宋德业
Owner THE SECOND XIANGYA HOSPITAL OF CENT SOUTH UNIV
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