Blood serum minuteness ribonucleic acid reagent kit and application in early diagnosis of hepatitis B

A technology of microRNA and kits, which can be used in the measurement/testing of microorganisms, biochemical equipment and methods, etc., and can solve rough and cumbersome problems

Inactive Publication Date: 2009-02-18
NANJING UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, traditional biochemical and molecular biological methods for clinical diagnosis of hepatitis B mainly include reverse passive hemagglutination, enzyme-linked immunosorbent assay, and genetic diagnosis. There are various deficiencies or deficiencies in efficie...

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0042] Example 1 Solexa sequencing of microRNA in serum / plasma of normal people, hepatitis B carriers, and hepatitis B patients, the specific steps are:

[0043] (1) Collect serum / plasma samples: collect several serum samples from normal people, hepatitis B carriers, and hepatitis B patients, and take a part of the serum from each normal person to make 40ml of mixed serum for the extraction of total RNA. The same is true for hepatitis B carriers and hepatitis B patients.

[0044] (2) Extract serum / plasma total RNA by Trizol reagent:

[0045] a Add 1ml of Trizol to every 1ml of serum (solexa needs about 40ml of serum), shake and mix by hand;

[0046] b Phase separation: Incubate the homogenate at 15-30°C for 5 minutes to fully dissociate the nucleoprotein complex, 1ml / 200ul chloroform, cover tightly, shake vigorously for 15 seconds, incubate at 15-30°C for 2-3 minutes, 2- Centrifuge at 10,000rcf (132,000rpm) at 8°C (4°C) for 15 minutes. After centrifugation, the mixture is di...

Embodiment 2

[0065] Example 2 Real-time PCR experiment of differentially expressed microRNAs in normal people, hepatitis B carriers, and hepatitis B patients' serum / plasma

[0066] The experimental steps of quantitative PCR are as follows:

[0067] (1) Extract the subject's serum / plasma total RNA, and obtain cDNA samples through RNA reverse transcription reaction; or collect the subject's serum / plasma samples, and use serum / plasma as a buffer to perform reverse transcription reaction to prepare cDNA samples;

[0068] (2) design primers with microribonucleic acid;

[0069] (3) adding fluorescent probes to carry out PCR reaction;

[0070] (4) Detect and compare the changes in the amount of microRNA in serum / plasma samples of normal people, hepatitis B carriers, and hepatitis B patients.

[0071] The dye used in the quantitative PCR experiment was EvaGreen, and the instrument used was ABI Prism 7500 fluorescent quantitative PCR instrument. The reaction conditions were 95°C, 5 minutes for 1 ...

Embodiment 3

[0079] Embodiment 3 is used for the making of the microRNA test kit of hepatitis B diagnosis and prediction

[0080] The production process and operation process of the microRNA kit are based on solexa sequencing technology, Realtime-PCR technology and biochip technology.

[0081] Firstly, the microRNA in normal human serum / plasma is determined by sequencing or PCR. Then, quantitative PCR technology and biochip technology are used to screen a type of serum / plasma microRNA with a large difference in expression between hepatitis B carriers, hepatitis B patients and normal people, as an indicator for predicting whether the disease occurs and diagnosing the degree of disease. Finally, the number of serum / plasma microRNAs corresponding to hepatitis B carriers and hepatitis B patients screened out is controlled at a dozen, which is the most optimized simplification made on the basis of the chip probe library. This kit includes a batch of serum / plasma microRNA primers, Taq enzyme, d...

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Abstract

invention discloses a kit for a serum micro-ribonucleic acid and the application in the early diagnosing of hepatitis B thereof which belong to the technical field of biotechnological pharmaceutics. The invention provides the combination of the micro-ribonucleic acid used for estimating the physiological and/or pathological states of a normal person, a HBVer and a patient with hepatitis B; the combination comprises all the micro-ribonucleic acids which have differential expression in the serum/plasm of the normal person, the HBVer and the patient with hepatitis B and prepares a diagnosing kit which can be used for the early diagnosing of hepatitis B. The combination and the method can be used for the early detection and nondirective therapy of hepatitis B; besides, the combination and the method have the advantages of high specificity, high efficiency, high sensitivity, low detection cost, convenient material obtaining, and the like. Besides, the samples are easily stored.

Description

a technical field [0001] The invention belongs to the technical field of biotechnology and pharmacy, and specifically relates to the application of serum / plasma microRNA in the diagnosis, prediction, evaluation of curative effect, screening of active ingredients of drugs, evaluation of drug effect and the like in hepatitis B. Two background technology [0002] Hepatitis B virus (HBV) is one of the most common pathogenic microorganisms in the world. According to statistics, there are currently about 400 million hepatitis B virus (HBV) carriers in the world. Chronic hepatitis B may lead to serious complications (such as liver cirrhosis and liver cancer). Statistics show that about 80% of primary liver cancers in the world are related to hepatitis B virus infection. Epidemiological surveys show that about 130 million people in my country have been carrying hepatitis B virus for a long time. Experts estimate that there are more than 20 million chronic hepatitis B patients in m...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/706
Inventor 曾科李丽民张辰宇陈熹白蕊卞桢张峻峰陈江宁
Owner NANJING UNIV
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