Rapid diagnostic kit based on loop-mediated isothermal amplification technique for hepatitis A virus genes and detection method thereof

A hepatitis A virus, ring-mediated isothermal technology, applied in the direction of microbial-based methods, resistance to vector-borne diseases, microbial measurement/testing, etc., can solve the problem of genetic rapid diagnostic kits for Mycobacterium tuberculosis , to achieve the effects of significant color difference, high yield, and efficient amplification

Active Publication Date: 2010-03-03
GUANGZHOU HUAFENG BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, isothermal amplification (Isothermal Amplification) nucleic acid rapid detection technology is a great progress in pathogenic nucleic acid detection technology. The established loop-mediated isotherma

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] Preparation of Example 1 Kit

[0039] (1) DNA synthesizer synthesizes the following oligodeoxynucleic acid primers:

[0040] Outer primer F3, the nucleotide sequence of which is shown in SEQ ID NO: 1;

[0041] Outer primer B3, the nucleotide sequence of which is shown in SEQ ID NO: 2;

[0042] The inner primer FIP, the nucleotide sequence of which is shown in SEQ ID NO: 3;

[0043]The nucleotide sequence of the inner primer BIP is shown in SEQ ID NO:4.

[0044] (2) Purchase DNA polymerase: Bst DNA polymerase is placed in a container.

[0045] (3) Preparation of reaction solution: the reaction solution contains 2mmol / L dNTP, 25mmol / L Tris-Cl, 12.5mmol / L potassium chloride, 12.5mmol / L ammonium sulfate, 10mmol / L magnesium sulfate, 0.125% by volume TritonX-100, 1mol / L betaine, 2 mol / L each of the inner primers FIP / BIP and 0.25 mol / L each of the outer primers F3 / B3, placed in a container.

[0046] (4) Purchase reverse transcriptase: AMV reverse transcriptase is placed ...

Embodiment 2

[0062] Example 2 Preparation of the kit

[0063] The formula of the reaction solution is: the reaction solution contains 1.6 mmol / L dNTP, 20 mmol / L Tris-Cl, 10 mmol / L potassium chloride, 10 mmol / L ammonium sulfate, 8 mmol / L magnesium sulfate, 0.1 vol% TritonX-100, 0.8 mol / L L betaine, 1.6 mol / L each of inner primers FIP / BIP and 0.2 mol / L each of outer primers F3 / B3.

[0064] The developer solution is EvaGreen.

[0065] Others are the same as in Example 1.

Embodiment 3

[0066] Example 3 Application of Hepatitis A Virus Gene Rapid Diagnosis Kit

[0067] Samples were collected according to the method in Section 2.1 of GB / T 18936-2003, 175 mL of glycine buffer (pH 7.5, glycine 0.5 mol / L, NaCl 0.3 mol / L) was added to 25 g of shellfish samples, mixed well with a stirrer, 37°C 30min. 4 ℃ 1000r / m20min, take the supernatant. The virus was precipitated overnight at 4°C with 8% PEG8000 (with a final concentration of 0.75 mol / L NaCl). 10000r / m15min, discard the supernatant, resuspend the pellet with 10mL PBS buffer (pH7.4), add an equal volume of 3-chloro-3-fluoro-ethane, mix well, 10000r / m15min at 4℃, carefully aspirate the supernatant, The virus was sedimented with 8% PEG8000 (with a final concentration of 0.75 mol / L NaCl) at 4°C overnight. 14000r / m15min at 4℃, discard the supernatant, and resuspend the pellet in an appropriate amount of PBS buffer. Take 1 / 4 volume of the suspension, extract once with an equal volume of chloroform, and carefully c...

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Abstract

The invention discloses a rapid diagnostic kit based on a loop-mediated isothermal amplification technique for hepatitis A virus genes and a detection method thereof. The kit comprises two pairs of primers, Bst DNA polymerase, revertase, an RNase inhibitor, a stabilizing solution, a reaction solution, a chromogenic solution and a positive contrast solution, wherein the nucleotide sequences of thetwo pairs of primers are shown in SEQ ID NO: 1-4; and the eight solutions are respectively contained in containers. The kit and the detection method can detect the hepatitis A virus with high efficiency and high specificity, are based on the loop-mediated isothermal amplification technique, apply six segments, four primers and one constant temperature to complete an amplification reaction within less than one hour, and have the advantages of low detection cost, short time consumption, high yield, high specificity, significant chromogenic difference between a positive result and a negative result, high authentication rate, distinctness and reliability.

Description

technical field [0001] The invention relates to biological detection reagents, in particular to a hepatitis A virus gene rapid diagnosis kit based on loop-mediated isothermal amplification technology and a detection method thereof. Background technique [0002] At present, there are various detection methods for hepatitis A virus, ranging from the national standard (GB / T 18936-2003) based on the isolation and identification of pathogenic microorganisms, morphological identification and serological identification, to the immunological detection technology of specific protein, nucleic acid Molecular biological detection methods such as probes and polymerase chain reaction (PCR) technology (GB / T 22287-2008). Among them, the detection of pathogenic nucleic acid has been greatly improved in terms of rapidity, safety, accuracy and sensitivity. These new technologies try to break through the old modes of microbiological detection such as traditional morphology and biochemical react...

Claims

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Application Information

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IPC IPC(8): C12Q1/70C12Q1/68C12R1/93
CPCY02A50/30
Inventor 曹以诚黄逸男李志勇杜正平陈洵谭惠媚王志强
Owner GUANGZHOU HUAFENG BIOTECH
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