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Liver cancer diagnostic reagent

A technology of liver cancer and reagents, which is applied in the field of reagents and kits for diagnosing liver cancer, achieving the effects of reducing medical burden, high accuracy, and easy mastery

Pending Publication Date: 2016-01-27
THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether salivary lncRNA and miRNA can be used as biomarkers to assist in the diagnosis of liver cancer has not yet been reported.

Method used

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  • Liver cancer diagnostic reagent
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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 saliva collection

[0037] Before the saliva collection, the study individuals were required to fast for more than 2 hours from eating, drinking, smoking and oral cleaning.

[0038]In order to increase the amount of saliva collected, use 2% citric acid solution to moisten the cotton tip of a sterile cotton swab, and then put the cotton tip on the rear side wall of one side of the individual's tongue for about 5 seconds, spit out the saliva, and then put the cotton swab on the other side The posterior wall of the tongue. so repeatedly collected. The amount of saliva collected should be more than 5ml, and the collection tube should be a 50ml sterile enzyme-free centrifuge tube. Centrifuge at 4°C, 3000g for 15min, take the supernatant into a 1.5ml Eppendoff tube, then centrifuge at 4°C, 12000g for 10min, then discard the precipitate and take the supernatant. All specimens were stored at -80°C after processing.

Embodiment 2

[0039] The extraction of embodiment 2 saliva total RNA

[0040] 1. Divide 1ml of saliva evenly on two 1.5ml enzyme-free EP tubes, that is, each EP tube contains 500μl of saliva, then add 15μl of proteinase K (20mg / ml) to each EP tube, mix well , and incubated overnight in a 65°C water bath. Because saliva contains more proteins such as digestive enzymes, which affects the effect of subsequent extraction of salivary RNA, this method can remove the influence of salivary proteins on the experiment.

[0041] 2. Add 0.5ml of acid-phenol-chloroform mixture (the main components are hydrochloric acid, phenol and chloroform) to each of the two EP tubes into the above-mentioned saliva mixture.

[0042] Configuration of acid phenol chloroform

[0043] 2.1 The ratio of phenol to chloroform is: 5:1

[0044] 2.2 Use concentrated hydrochloric acid to adjust the pH value, and set the pH value at about 4.5, which is most conducive to the extraction of RNA. Mix well.

[0045] 3. Manually s...

Embodiment 3

[0057] Example 3 Reverse transcription and expression detection of salivary H19, miR-21 and miR-222

[0058] 1. Reverse transcription reaction of salivary H19

[0059] Take 2 μL RNA for reverse transcription. After reacting at 65°C for 5 minutes, immediately ice-bath for 2 minutes.

[0060] Add 2 μl of 5×RTBuffer, 0.5 μl of EnzymeMix, 0.5 μl of PrimerMix (both products of Toyobo, Cat. No. FSK-100) and 5 μl of nuclease-free water treated with DEPC to the above 2 μL of RNA, and then proceed with reverse transcription at 37°C for 15 minutes , 98°C 5min, 4°C∞.

[0061] 2. Reverse transcription reaction of salivary miR-21 and miR-222

[0062] Take 2 μL RNA for reverse transcription. Add 2 μL of RTPrimer [Ribo Company, Cat. No. miRQ0000076-1-2 (miR-21) or miRQ0004569-1-2 (miR-222)] and 7 μL of nuclease-free water to each 2 μL of RNA. Immediately after reacting at 70°C for 10 minutes, ice bath for 2 minutes. Add 5 μL RTbuffer (TAKARA company, product number 2641A), 2 μL dNTP (T...

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Abstract

The present invention belongs to the field of biomedicines, and relates to a liver cancer diagnostic reagent and a kit. The liver cancer diagnostic reagent is a reagent for detection of a long-chain non-coding RNA (long non-coding RNA, lncRNA) H19 and the two kinds of micro RNA (microRNA, miRNA) miR-21 and miR-222, and a detection target of the reagent is a saliva sample. The liver cancer detection kit using saliva as a sample contains protease and the H19, miR-21 and miR-222 detection reagent. For the first time, biomarkers sensitive and specific to liver cancer diagnosis are found in the saliva, and the liver cancer diagnostic reagent has great significance to early diagnosis and treatment of liver cancer and reduction of mortality and the medical burden in China.

Description

technical field [0001] The invention belongs to the field of biomedicine and relates to a reagent and a kit for diagnosing liver cancer. Background technique [0002] my country is a big country with liver cancer, and the incidence and mortality of liver cancer account for more than half of the world's total. The accurate diagnosis of liver cancer has important practical significance. [0003] Currently, serum AFP (alpha-fetoprotein) is commonly used as a biomarker for screening liver cancer in the world. But its sensitivity is only 39% to 65%, and its specificity is only 76% to 94%. Because of its unsatisfactory sensitivity and specificity for detecting liver cancer, the treatment guidelines in the United States do not recommend serum AFP for the diagnosis of liver cancer. Both Chinese and European treatment guidelines point out that the AFP level of normal individuals should be less than 20ng / ml, and greater than 400ng / ml can indicate the diagnosis of liver cancer. And...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q1/6851C12Q2600/178C12Q2531/113
Inventor 吴斌
Owner THE THIRD AFFILIATED HOSPITAL OF SUN YAT SEN UNIV
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