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Hepatitis C virus RNA (ribonucleic acid) extracting kit

A hepatitis C virus and nucleic acid extraction reagent technology, which is applied in the field of biomedical detection, can solve the problems of long window period and long-term existence, and achieve the effect of high safety, efficient extraction and improved stability

Active Publication Date: 2017-10-17
SICHUAN MACCURA BIOTECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Immunological approach has its own limitations: long window period (50-70 days), and only represents past infection, can persist long after viral clearance
In addition, the classic RNA extraction methods often use toxic reagents such as guanidine isothiocyanate, phenol, chloroform, etc., which is also one of its inevitable inherent defects

Method used

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  • Hepatitis C virus RNA (ribonucleic acid) extracting kit
  • Hepatitis C virus RNA (ribonucleic acid) extracting kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Preparation of Plasma HCV Extraction Reagent

[0038] The following four formulations were prepared for the extraction of plasma HCV.

[0039] Formulation A: kit of the present invention

[0040] Nucleic acid extraction reagent 1: proteinase K 20mg / mL;

[0041] Nucleic acid extraction reagent 2 (lysate): guanidine hydrochloride 573.18g / L, triton (TX-100) 200ml / L, citric acid 1.68g / L, sodium citrate 7.35g / L, pH 4.4±0.1;

[0042] Nucleic acid extraction reagent 3: magnetic beads, 100%;

[0043] Nucleic acid extraction reagent 4 (washing liquid I): Guanidine hydrochloride 382.12g / L, citric acid 1.05g / L, sodium citrate 11.76g / L, isopropanol 400mL / L, pH is 5.4 ± 0.1;

[0044] Nucleic acid extraction reagent 5 (washing solution II): sodium chloride 5.84g / L, Triton (TX-100) 10ml / L;

[0045] Nucleic acid extraction reagent 6: mineral oil 100% (v / v);

[0046] Nucleic acid extraction reagent 7: 1MTris (pH 8.0) 10ml / L, 0.5M EDTA (pH 8.0) 2mL / L.

[0047] During the preparatio...

Embodiment 2

[0063] Comparison of Extraction Efficiency of Different Formulas

[0064] HCV plasma samples with a concentration of 5E5 IU / mL were used, and various formulations in Example 1 were used to perform RNA extraction experiments respectively.

[0065] The extraction methods corresponding to formula A and formula E are as follows:

[0066] a. Take 1.5mL centrifuge tubes and mark them separately, and add 10 μL of nucleic acid extraction reagent 1 to each tube;

[0067] b. Add 200 μL of the sample to be tested in each tube, cover the tube cap, shake and mix for 5 seconds, and centrifuge briefly;

[0068] c. Add 600 μL of nucleic acid extraction reagent 2 and 5 μL of nucleic acid extraction reagent 3 to each tube, cover the tube, shake and mix for 10 seconds, and let stand at room temperature for 10 minutes;

[0069] d. Instant centrifugation, place the centrifuge tube on a magnetic separator, and slowly suck out the solution after 3 minutes (be careful not to touch the brown matter ...

Embodiment 3

[0081] Extraction sensitivity and stability testing

[0082] The HCV-positive plasma sample E1 was selected, and a part thereof was taken out in triplicate for 10-fold dilution to obtain 10-fold diluted samples E2, E3, and E4, respectively.

[0083] The nucleic acid concentrations of samples E1, E2, E3 and E4 were measured using the formula B (Sunshine Bio HCV Nucleic Acid Quantitative Detection Kit) described in Example 1 to obtain the data in Table 2.

[0084] Table 2: Formulation B measured values

[0085]

E1

E2

E3

E4

Ct value

29.75

33.11

35.95

40

Concentration (IU / mL)

2.55E+04

2.66E+03

393.43

22.57

[0086] According to Table 2, it can be seen that the concentration calculated by formula B measuring sample E4 is 22.57IU / mL, which is lower than the lower sensitivity limit of formula B 25IU / mL.

[0087] Dilute the E4 sample 2-fold to obtain 1 / 2 E4 sample with a theoretical concentration of 11IU / mL. ...

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Abstract

The invention relates to a hepatitis C virus RNA extracting kit, belongs to the field of biomedical detection and relates to the nucleic acid isolation technology, particularly to the hepatitis C virus RNA extracting technology. By combining guanidine hydrochloride with a citric acid buffer system, the hepatitis C virus RNA extracting kit achieves the technical effects of effectively extracting hepatitis C virus RNA in samples in high security, high sensitivity and high stability.

Description

technical field [0001] The invention belongs to the field of biomedical detection, and relates to nucleic acid purification technology, in particular to the technology of extracting hepatitis C virus RNA. Background technique [0002] Hepatitis C is viral hepatitis caused by hepatitis C virus (Hepatitis virus C, HCV) infection. Since there is no established preventive method for hepatitis C, and some patients with hepatitis C may develop liver cirrhosis or even liver cancer, early detection and early treatment of HCV infection are very important. [0003] Currently, there are mainly two types of HCV detection methods commonly used in clinic: immunological method for detecting anti-HCV or HCV encoded protein, and PCR method for detecting hepatitis C virus ribonucleic acid (HCV-RNA). The immunological approach has its own limitations: the window period is long (50-70 days), and it only represents past infection, which can persist long after the virus has cleared. HCV-RNA in ...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1013C12Q2527/125
Inventor 刘春晖杨文秀杨丽廖世玉李莹
Owner SICHUAN MACCURA BIOTECH CO LTD
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